The Effects of 3-Isobutyl-1-methylxanthine (IBMX) on Nuclear and Cytoplasmic Maturation of Porcine Oocytes In Vitro
Seong-Sung Kwaka, Seung-Hoon Janga, Se-Heon Jeong, Yubyeol Jeon, Dibyendu Biswas and Sang-Hwan Hyun* Laboratory of Veterinary Embryology and Biotechnology, Department of Veterinary Medicine,
College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Korea
ABSTRACT
The 3-isobutyl-1-methylxanthine (IBMX) is non-selective phosphodiesterase and is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte. The present study was conducted to analyze: (1) nuclear maturation (examined by the Hoechst staining), (2) whether cytoplasmic maturation (examined by the intracellular glutathione (GSH) concentration) of porcine oocytes is improved during meiotic arrest after pre- maturation (22 h) with IBMX. Before in vitro maturation (IVM), oocytes were treated with 1 mM IBMX for 22 h.
After 22 h of pre-maturation, the higher rate of IBMX treated group oocytes were arrested at the germinal vesicle (GV) stage (42.3%) than control IVM oocytes (10.1%). It appears that the effect of IBMX on the resumption of meiosis has shown clearly. In the end of IVM, the reversibility of the IBMX effect on the nuclear maturation has been corroborated in this study by the high proportions of MII stage oocytes (72.5%) reached after 44 h of IVM following the 22 h of inhibition. However, intracellular GSH concentrations were lower in the oocytes treated with IBMX than the control oocytes (6.78 and 12.94 pmol/oocyte, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pre-treated with IBMX for 22 h did not equal that of control oocytes in the current IVM system.
These results indicate that pre-maturation with IBMX for 22 h may not be beneficial in porcine IVM system.
(Key words : porcine oocytes, IBMX, phosphodiesterase, cAMP, resumption of meiosis, nuclear maturation)
†This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (#2012004885).
aThese authors contributed equally to this work.
*Correspondence : E-mail : [email protected]
INTRODUCTION
In the ovarian follicle, the oocyte is arrested at the end of the first meiotic prophase and remains in this static state for prolonged periods of time, up to 50 years in the human. Soon after the preovulatory LH surge the meiotic arrest is released and oocytes resume the first meiotic division which culminates with the extrusion of the first polar body. Maturing oocytes continue with the second meiotic division and are again arrested in metaphase of meiosis II, which will be completed only after fertilization (Tsafriri and Dekel, 1994). Resumption of meiosis is characterized by chromatin condensation, disruption of the nuclear envelope (germinal vesicle breakdown, GVBD), forma- tion of the microtubule spindle and separation of homologous chromosomes. This process, also known as oocyte maturation, is indispensable for fertilization as immature oocytes cannot be penetrated and activated by the sperm (Tsafriri and Dekel, 1994).
It is well established that the cyclic nucleotide cAMP plays a key role in the control of oocyte maturation in mammals (Tsafriri and Dekel, 1994), in amphibians (Maller et al., 1979) and some invertebrates (Meijer et al., 1989). Inhibition of meio- tic resumption by cAMP in mammals was originally proposed by Cho and collaborators (Cho et al., 1974). Meiotic re- sumption is prevented by high cAMP levels following incu- bation with cAMP analogs, with pharmacological agents that stimulate cAMP production, or by inhibitors of cAMP degra- dation. Spontaneous resumption of meiosis is efficiently blocked by incubation with dibutyryl cAMP (Dekel et al., 1981), forskolin (Racowsky, 1984), and PDE inhibitors (Aktas et al., 1995; Thomas et al., 2002). There is also evidence indicating that a decrease in intra-oocyte cAMP precedes the breakdown of the germinal vesicle, at least in vitro (Schultz et al., 1983;
Vivarelli et al., 1983; Aberdam et al., 1987). Thus, high levels of cAMP within the oocyte maintain meiotic arrest, whereas a decrease in this cyclic nucleotide is a signal necessary for
oocyte maturation.
The overall lower developmental capacity of oocytes ma- tured in vitro is believed to be due to suboptimal oocyte development in vitro and asynchronous nuclear and cytoplas- mic maturation (Eppig et al., 1994). Numerous studies suggest that successful fertilization and embryonic development depend not only on nuclear maturity of the oocyte but also on cyto- plasmic maturation. Temporary blockage and delaying of spon- taneous nuclear maturation might improve the synchronization of nuclear and cytoplasmic maturation during in vitro culture of immature oocytes and ultimately improve the developmental competence of IVM oocytes (Anderiesz et al., 2000). Along this line of thinking, several pharmacologic approaches have been accomplished to postpone resumption of meiosis and allow cytoplasmic maturation in vitro (Faerge et al., 2001;
Guixue et al., 2001; Sirard, 2001; Wu et al., 2002; Le Beux et al., 2003; Shimada et al., 2003).
A number of methylxanthines, including caffeine and theo- phylline, inhibit adenosine 3',5'-cyclic monophosphate phospho- diesterase (cAMP PDE). IBMX has been shown to be a potent inhibitor of cAMP PDE, significantly more effective than theo- phylline. IBMX inhibits cyclic nucleotide PDE with subsequent inhibition of cyclic nucleotide hydrolysis, resulting in accumu- lation of cyclic AMP and guanosine 3',5'-cyclic monophosphate.
In a study of cAMP and insulin released by islets of Langer- hans, IBMX at 1 mM caused a marked increase in the intra- cellular concentration of cAMP in the presence of glucose.
To examine whether cytoplasmic maturation of porcine oocytes is improved during meiotic arrest, the effects of IBMX on the concentration of intracellular GSH and the kinetics of nuclear maturation were investigated.
MATERIALS AND METHODS
1. Chemicals
Unless otherwise indicated, all chemicals and reagents were purchased from Sigma Aldrich Co. (St. Louis, MO. USA).
2. Oocyte Collection and In Vitro Maturation
Porcine ovaries were obtained from prepubertal gilts at local slaughterhouse and transported to the laboratory within 2 hours in physiological saline supplemented with 100 IU/ml peni- cillin G and 100 mg/l streptomycin sulfate at 32 to 35℃. The cumulus oocyte complexes (COCs) were aspirated using an 18-gauge needle attached to a 10 ml disposable syringe from
superficial follicles 3 to 6 mm in diameter and pooled in to 15 ml conical tubes and allowed at 37℃ during 5 min to settle down as sediment. The supernatant was discarded and the pre- cipitate was resuspended with HEPES-buffered Tyrode’s me- dium (TLH) containing 0.05% (w/v) polyvinyl alcohol (TLH- PVA) and observed under a stereomicroscope. Only compact COCs with ³ 3 uniform layers of compact cumulus cells and homogenous cytoplasm were recovered from the collected fluid and washed three times in TLH-PVA. Approximately 50～60 COCs were transferred into each well of a 4-well Nunc dish (Nunc, Roskilde, Denmark) containing 500 μl of culture medium (TCM-199; Invitrogen Corporation, Carlsbad, CA, USA) which was supplemented with 0.6 mM cysteine, 0.91 mM sodium py- ruvate, 10 ng/ml epidermal growth factor, 75 μg/ml kanamy- cin, 1 μg/ml insulin, 10% (v/v) porcine follicular fluid (pFF), 10 IU/ml equine Chronic Gonadotropin (eCG), and 10 IU/ml hCG (Intervet, Boxmeer, Netherland). For IVM, the selected COCs were incubated at 39℃ in a humidified atmosphere of 5% CO2 in 95% humidified air. After 22 h of maturation with hormones, the COCs were washed two times in hormone-free IVM medium and then cultured in hormone-free IVM medium for an additional 22 h. The COCs were treated with or without 1 mM IBMX before IVM (pre-maturation), according to the experimental design.
3. Assessment of Nuclear Maturation
After 0, 22, 44 and 66 h of oocytes maturation, oocytes were stained with 10μg/ml Hoechst 33342 in absolute alcohol, visualized under epifluorescence microscopy (330～385 nm; at a magnification of 400×) and assessed for nuclear maturation.
Oocyte nuclear maturation status was classified as (1) GV stage oocyte when displaying a germinal vesicle, (2) intermediate stage oocyte when displaying germinal vesicle breakdown and condensed chromatin or metaphase I organized chromatin, or (3) mature stage oocyte when displaying anaphase I, telophase I or metaphase II DNA configuration (Fig. 1).
4. Intracellular GSH Assay
After IVM (42–44 hr), the oocytes were stripped of surroun- ding cumulus cells by repeated pipetting, and matured oocytes (defined as oocytes in which the first PB was visualized under a stereomicroscope) were selected for GSH measurement.
Intracellular GSH was measured as described by (Baker et al., 1990) with some modification. Briefly, MII oocytes from each group were washed three times in 0.2 M sodium phosphate
Fig. 1. Photomicrographs of porcine oocytes stained with Hoechst 33342. (A) GV stage oocyte (displaying a germinal vesicle and decondensed chromatin). (B) M-I stage oocyte (dis- playing germinal vesicle breakdown and condensed chro- matin). (C) M-II stage oocyte (nucleus and 1st polar body (arrow) are seen in ooplasm), Original magnification. 400x, Scale bar=50μm.
buffer (Na2HPO4, NaH2PO4, and 10 mM EDTA-2Na, pH 7.2), and groups of 50～60 oocytes (per sample) in 10 μl sodium phosphate buffer were transferred to 1.7-ml microfuge tubes;
10 μl of 1.25 mM phosphoric acid (final concentration of 0.625M H3PO4) in distilled water was added to each sample.
Tubes containing the samples were frozen at 80℃ until analysis.
GSH concentrations in the oocytes were determined using a 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB)-GSH reductase (GSSG) recycling assay. Before the assay, the frozen samples were thawed at room temperature, vortexed, centrifuged, and microscopically evaluated to ensure complete lysis of the oocytes.
The supernatants were transferred to a 96-well microtiter plate and, for each sample, 700 μl of 0.33 mg/ml NADPH in 0.2 M assay buffer containing 10 mM EDTA (stock buffer, pH 7.2), 100 μl of 6 mM DTNB in the stock buffer, and 180 μl of distilled water and 1 U per sample of GSSG (Sigma G3664, 441 U/ml) were added in a conical tube, mixed, and imme- diately added to the sample. The plate was immediately placed in a microtiter plate reader, and optical density was measured with a 405-nm filter (Emax, Molecular Devices, Sunnyvale, CA, USA). The formation of 5-thio-2 nitrobenzoic acid was monitored every 30 sec for 3 min. Standard curves were pre- pared for each assay, and GSH content per sample was deter- mined by the standard curve. The GSH concentrations (pmol/
oocyte) were calculated by dividing the total concentration per sample by the total number of oocytes present in the sample.
5. Experimental Design
The experimental design is schematically represented in Fig. 2.
Experiment 1. Nuclear status after 22 h pre-maturation with IBMX.
This experiment was conducted to investigate whether pre-
Experiment 1. Nuclear Status After 22 h Culture in 1 mM IBMX
Experiment 2. Nuclear Progression After IVM With a Prema- turation Period in IBMX
Experiment 3. Oocyte Glutathione Content After IBMX Treatment
Fig. 2. Experimental design.
maturation of oocytes with IBMX for the first 22 h before IVM delay the nuclear maturation. After maturation, oocytes nuclear status (GV to MII) was recorded in all groups. This experiment was performed in four replicates.
Experiment 2. Nuclear maturation and cumulus cell expansion after IVM in pre-maturation with IBMX.
In order to assess the reversibility of the treatment with IBMX, COCs were IVM for 44 and 66 h. In four replicates, COCs were collected and allotted to four groups: (1) normal, control IVM group (A grade oocytes); (2) normal, control IVM group (B grade oocytes); (3) treated with IBMX in 22 h (pre-maturation) and thereafter IVM for 22 h without IBMX (B grade); (4) treated with IBMX for 22 h (pre-maturation) and thereafter IVM for 44 h without IBMX (B grade).
Experiment 3. Intracellular GSH concentrations after IBMX treatment.
This experiment was designed to examine the influence of IBMX on the intracellular GSH content of oocytes after matu- ration with or without IBMX in 44 and 66 h. The experimen-
tal group is same as experiment 2. GSH content was measured as described above.
6. Statistical Analysis
The statistical analysis was conducted using SPSS Inc. soft- ware (PASW Statistics 17). A one-way analysis of variance with Duncan multiple-range test was used to assess maturation rates, total GSH levels. Data were presented as mean ± SEM.
Differences were considered to be significant if the P value was less than 0.05.
RESULTS
Data for nuclear status during IVM are shown in Table 1 and Table 2. IBMX was effective in inhibiting the meiotic resumption after 22 h of pre-maturation, giving a significantly (p<0.05) higher percentage of GV stage oocytes in the IBMX- treated group than in control IVM group (Table 1). After 44 and 66 h IVM, oocytes from the IBMX-treated group resumed meiosis and reached MII in the same proportions as in the control IVM group (P>0.05) (Table 2). The expansion for cumulus cells are shown in Fig. 3. After 22 h of maturation, cumulus cells expansion of IBMX treated oocytes were inhibited,
Table 1. Nuclear status of porcine oocytes after 22 h in vitro maturation with or without 1 mM IBMX treatment
Group No. of oocytes examined GV (%) M I (%) M II (%)
Before culture (0 h) 135 135 (100 ± 0)a 0 (0)c 0 (0)c
IVM (22 h) - A' 97 16 (17.3 ± 3.7)b 75 (77.6 ± 1.3)a 6 (5.1 ± 5.1)a,b
IVM (22 h) - B' 127 13 (10.1 ± 3.1)b 93 (73.3 ± 0.6)a 21 (16.6 ± 3.6)a
IBMX (22 h) - B' 159 65 (42.3 ± 8.0)c 81 (50.6 ± 6.1)b 13 (7.1 ± 2.4)a,b
a～c Values with different superscripts within same column are significantly different (p<0.05). The data are mean ± SEM.
GV: Germinal vesicle stage. MI: Metaphase I stage. MII: Metaphase II stage.
Table 2. Nuclear status of porcine oocytes after in vitro maturation with or without IBMX treatment during pre-maturation period
Group No. of oocytes examined GV (%) M I (%) M II (%)
IVM (44 h) - A' 99 6 (6.1 ± 0.7) 8 (8.6 ± 5.0)a 85 (85.2 ± 5.7)
IVM (44 h) - B' 389 26 (6.9 ± 1.7) 65 (18.4 ± 3.6)a,b 298 (74.8 ± 4.3)
IBMX (22 h) + IVM (22 h) - B' 297 25 (8.3 ± 2.1) 68 (22.8 ± 3.2)b 204 (68.9 ± 4.2) IBMX (22 h) + IVM (44 h) - B' 291 19 (6.2 ± 1.8) 61 (21.3 ± 4.0)a,b 211 (72.5 ± 4.7)
a,b Values with different superscripts within same column are significantly different (p<0.05). The data are mean ± SEM.
GV: Germinal vesicle stage. MI: Metaphase I stage. MII: Metaphase II stage.
Fig. 3. Time dependent morphological changes of cumulus oocyte complexes (COCs) in each group. Expansion of cumulus cell is inhibited in 22 h culture of IBMX. In 44 h and 66 h of IBMX culture, the cumulus cell expansion is similar to the control.
while cumulus cells expansion of grade B showed less than that of grade A cumulus cells. After 44 h of maturation, expan- sion of cumulus cells of IBMX treated was less than control groups but after more 22 h maturation (66 h IVM), it was as same as 44 h IVM of grade B control group. The data for
intracellular GSH concentration are presented in Fig. 4. In- tracellular GSH concentration in 22 h pre-maturation with IBMX was significantly decreased compared with control of grade A but there were no significant difference between IBMX group and control of grade B (Fig. 4).
DISCUSSION
The effects of IBMX on the resumption of meiosis have been shown previously in bovine oocytes (Barretto et al., 2007).
Meiotic inhibitors such as IBMX and roscovitine delay the progression of nuclear maturation in bovine oocytes. After 22 h of pre-maturation, a percentage of oocytes treated with IBMX were blocked at the GV stage was significantly high in com- parison with non-treated oocytes. The result of nuclear status of porcine oocytes after 22 h pre-maturation with or without 1 mM IBMX treatment is indicates that IBMX effectively blocks the resumption of meiosis as we expected.
Nuclear maturation of porcine oocytes after in vitro matu- ration with (IBMX-IVM) or without (IVM) a pre-maturation for 22 h in 1 mM IBMX was assessed to examine whether oocyte treated with IBMX is reversed. The reversibility of the IBMX effect on the nuclear maturation has been corroborated in this study by the high proportions of MII stage oocytes reached after 44 h and 66 h of IVM (68.9% and 72.5%, res- pectively). This result is similar to that of control group (74.8%).
The poor developmental competence in porcine oocytes ma- tured in vitro may be caused by inappropriate cytoplasmic ma-
Fig. 4. Intracellular GSH content in porcine oocytes with normal IVM and IBMX Treated group. a,b Values with different superscripts within same column are significantly different (p<0.05). The data are mean ± SEM.
turation, even though nuclear maturation is completed (Mar- chal et al., 2003). To date, porcine IVM/IVP techniques have progressed and are well studied (Nagai, 2001), but the poten- tial to develop transferable embryos from porcine oocytes ma- tured in vitro (Wang et al., 1997) remains low compared to those matured in vivo (Wang et al., 1999). Therefore, pre-ma- turation of the oocytes in the presence of meiotic inhibitors (e.g. IBMX) could more precisely mimic in vivo oocyte capaci- tation by using this procedure, the oocytes could undergo the repositioning of organelles and storing of newly synthesized proteins could occur that could improve its cytoplasmic matu- ration and also developmental competence. To analyze the effect of IBMX on cytoplasmic maturation, intracellular GSH concen- tration was evaluated. Intracellular GSH is a molecular marker in mature oocytes that predicts cytoplasmic maturation in porcine oocytes (Abeydeera et al., 1998) and is involved in va- rious cellular processes, including DNA and protein synthesis, metabolism of chemicals, cellular protection, and amino acid transport.
In the present study, however, no beneficial effect of pre- maturation with IBMX was observed in cytoplasmic matura- tion of porcine oocytes. Pre-maturation with IBMX at concen- trations of 1 mM for 22 hours seems to be rather harmful to cytoplasmic maturation of porcine oocytes, as shown by intra- cellular GSH analysis. The present study shows that oocytes pre-maturation with IBMX for 22 h had a lower level of intracellular GSH than control (Fig. 4).
In theory, short-term meiotic arrest of immature oocytes be- fore beginning IVM might be helpful because delaying sponta- neous IVM would permit more complete cytoplasmic matura- tion. Although increased developmental competences of oocytes after meiotic arrest for 24 h were reported in bovine studies (Ponderato et al., 2002; Kaedei et al., 2010), the same bene- ficial effect was not observed in the present study. Possible reason may be due to the long time of porcine IVM (22 h pre-maturation + 44 h IVM) than bovine IVM (commonly 24 h). Because of long time maturation in vitro made the oocytes get more oxidative stress, so the concentration of intracellular GSH might be decreased than the control. In addition, intra- cellular GSH might be decreased due to the harmful effect of IBMX on cumulus cells which are involved in GSH produc- tion (Fig. 3).
In conclusion, these results demonstrated that the meiotic inhibitors IBMX delay the progression of nuclear maturation.
Nuclear maturation of IBMX treated oocytes had no diffe-
rences with control IVM oocytes, but the cytoplasmic matura- tion assessed by the analysis of intracellular GSH concentra- tion was decreased. Therefore, pre-maturation with IBMX for 22 h may not be beneficial in porcine IVM system.
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(접수: 2012. 8. 19 / 심사: 2012. 8. 20 / 채택: 2012. 9. 5)
