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Developmental capacity of bovine cumulus oocyte complexes after transcriptional inhibition of germinal vesicle breakdown
Theriogenology 61 (2004) 1499–1511
Developmental capacity of bovine cumulus oocyte complexes after transcriptional inhibition of germinal vesicle breakdown K.F. Rodriguez, C.E. Farin* Department of Animal Science, North Carolina State University, Box 7621, 231 B Polk Hall, Raleigh, NC 27695-7621, USA Received 12 November 2002; accepted 28 August 2003
Abstract Oocytes cultured in the presence of FSH and the transcriptional inhibitor, 5,6-dichloro-1-b-Dribofuranosylbenzimidazole (DRB), remain in meiotic arrest at the germinal vesicle (GV) stage. The objectives of this study were to assess the kinetics of maturation and the developmental capacity of bovine cumulus oocyte complexes (COC) following release from prolonged meiotic arrest by DRB. In Experiment I, COC were cultured for 20 h in Tissue culture medium (TCM)-199 supplemented with 10% estrus cow serum (ECS), 5 mg/ml FSH and 1 mg/ml estradiol in the presence of 120 mM DRB. COC were then released from meiotic arrest and cultured for 20 h in DRB-free medium. Control COC were cultured for 20 h in DRB-free medium, with culture initiated concomitant to the release of DRB-treated COC from meiotic arrest. Nuclear maturation was assessed after 0, 5, 10, 15, and 20 h of culture in DRB-free medium. The proportion of DRB-arrested oocytes reaching metaphase II (MII) following 20 h culture in DRB-free medium was not significantly different from controls (96 4% versus 99 4%). In Experiment II, COC were cultured for 20 h in TCM-199 supplemented with 10% ECS, 10 mg/ml LH, 5 mg/ml FSH, and 1 mg/ml estradiol in the presence or absence of 120 mM DRB. COC in the DRB-treated group were then washed and matured coincident with a second group of control COC for 20 h in DRB-free medium. COC in both groups were fertilized and then randomly assigned to one of two culture systems: TCM-199 þ 10%ECS or mSOF þ 0:6% fatty acid-free BSA. Development was assessed at 72 h post insemination (hpi), 168 hpi (Day 7) and 216 hpi (Day 9). In this experiment, culture with DRB-arrested oocyte maturation at the GV stage (DRB, 85 3% GV; Control, 2 3% GV; P < 0:001). Following release from arrest, maturation and fertilization, the proportion of COC that cleaved by 72 hpi was decreased by treatment with DRB (DRB: 78 3% versus Control: 90 3%; P < 0:05). However, no effect of DRB was found on the proportion of cleaved zygotes that reached the blastocyst stage on either Day 7 or Day 9 of culture (Day 7: DRB 16 2% versus Control, 21 2%; Day 9: DRB 23 3% versus Control, 31 3%). More embryos reached the blastocyst stage in the * Corresponding author. Tel.: þ1-919-515-4022; fax: þ1-919-515-7780. E-mail address: [email protected] (C.E. Farin).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.08.016
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TCM-199/serum culture system compared to the mSOF/BSA system on both Days 7 and 9 (Day 7: TCM-199, 23 2% versus mSOF, 13 2%, P < 0:05; Day 9: TCM-199, 32 3% versus mSOF, 22 3%, P < 0:05). In summary, bovine COC maintained in meiotic arrest for 20 h by culture in the presence of the transcriptional inhibitor DRB retained their capacity to develop to the blastocyst stage after fertilization in vitro. # 2003 Elsevier Inc. All rights reserved. Keywords: Oocyte; Meiosis; Transcription; Blastocyst; Bovine
1. Introduction Throughout follicular development, oocytes remain arrested at prophase I of meiosis. During this period of meiotic arrest, oocyte growth continues and maternal mRNA transcripts required for acquisition of developmental competence accumulate in the cytoplasm [1–3]. Both cytoplasmic and nuclear maturation must occur for the oocyte to become competent to support subsequent post-fertilization development. Cytoplasmic maturation includes not only the accumulation of maternal mRNA, but also cellular changes such as alignment of cortical granules [4], increased lipid accumulation [4] and accumulation of glutathione [5,6]. Nuclear maturation refers to acquisition of the ability to undergo dissolution of the germinal vesicle (nuclear membrane), condensation of the chromosomes, release of the first polar body, and subsequent arrest at metaphase II (MII) [7,8]. In vivo, resumption of meiosis is induced by the preovulatory surge of gonadotropins [9]. However, when bovine cumulus oocyte complexes (COC) are aspirated from antral follicles greater than 2 mm in diameter and cultured in vitro, they resume meiosis either spontaneously or in the presence of gonadotropins [10,11]. De novo transcription is required for gonadotropin-mediated, but not spontaneous, oocyte maturation [12]. Specific inhibitors of transcription, such as a-amanitin or 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB), have been used to arrest gonadotropin-mediated resumption of meiosis in the pig [13], sheep [14], cow [12,15], and mouse [16]. For transcriptional inhibitors to effectively arrest maturation at the germinal vesicle (GV) stage, several layers of cumulus cells must surround the oocytes and gonadotropins must be present in the maturation medium [12,16]. These observations support the conclusion that gonadotropin stimulation results in a transcriptional event that occurs in the cumulus cells that is required for initiation of GVBD [12,16]. Only 30–40% of bovine oocytes that undergo maturation and fertilization in vitro develop to the blastocyst stage [17–19]; in contrast, approximately 80% of oocytes matured and fertilized in vivo reach the blastocyst stage [20]. It has been suggested that the developmental competence of in vitro matured oocytes might be increased if oocytes could be maintained in meiotic arrest allowing an additional opportunity for cytoplasmic maturation to occur [3,21]. Roscovitine, a purine that specifically inhibits maturation promoting factor (MPF) activity and blocks cell cycle progression, has been used to maintain bovine oocytes at the GV stage without compromising their subsequent developmental potential [21]. In contrast, developmental capacity after fertilization of bovine COC maintained in transcriptional arrest of meiosis with a-amanitin is severely reduced [22]; this transcriptional inhibitor specifically blocks nucleoplasmic RNA synthesis by
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irreversibly binding to RNA polymerase II [23]. That the developmental capacity following a-amanitin exposure may be compromised is consistent with its mechanism of action. Alternatively, DRB specifically and reversibly blocks transcription by preventing the formation of a stable transcription initiation complex [24]. Because the action of DRB is highly reversible [24], it would be anticipated that oocyte developmental competence may be better maintained following meiotic arrest with this inhibitor. There were differences in the proportion of COC that reached the blastocyst stage, depending on the culture system utilized and specifically on the presence or absence of serum in the culture system [20,25,26]. Furthermore, in vitro culture systems can influence gene expression, morphology and blastocyst development [26–28]. Therefore, the developmental capacity of oocytes maintained in meiotic arrest by prolonged culture in the presence of a transcriptional inhibitor may also be influenced by culture conditions following fertilization. The objectives of this study were first, to analyze the kinetics of oocyte maturation following release from transcriptional inhibition of meiosis by DRB; and second, to assess the developmental capacity of bovine COC that were maintained in prolonged meiotic arrest with DRB using two different culture systems.
2. Materials and methods 2.1. Reagents and media Tissue culture medium (TCM-199 with Earl’s salts) was purchased from Gibco BRL (Grand Island, NY, USA). Equine pituitary LH (11.5 NIH LH-S1 U/mg), porcine pituitary FSH (50 mg/vial Armour FSH standard) and the transcriptional inhibitor DRB were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents and medium supplements were of tissue culture grade and purchased from Sigma Chemical Co. 2.2. Experiment I: kinetics of oocyte maturation following DRB arrest Ovaries were collected at a local abattoir and transported to the laboratory at room temperature in saline with 0.75 mg/ml penicillin. Approximately 1.5 h after ovary collection, follicles 2–8 mm in diameter were aspirated using an 18-gauge needle and 10 ml syringe. COC with several layers of cumulus cells were collected, washed five times in modified Tyrode’s medium (TL-Hepes), and cultured for 20 h in 1 ml of TCM-199, supplemented with 10% heat-inactivated estrus cow serum (ECS), 5 mg/ml FSH, 1 mg/ml estradiol, 200 mM pyruvate, and 50 mg/ml gentamicin in the presence of either DRB (120 mM in 0.2% DMSO) or vehicle control (0.2% DMSO). All maturation cultures were maintained at 39 8C in an atmosphere of 5% CO2 in air with 100% humidity. COC were cultured in the presence of DRB for a total of 20 h. The treatment medium was changed every 4 h throughout this period to maximally inhibit oocyte maturation [12]. At the end of the treatment period, COC were washed six times in maturation medium (TCM-199) supplemented with 10% heat-inactivated ECS, 10 mg/ml LH, 5 mg/ml FSH, 1 mg/ml estradiol, 200 mM pyruvate, and 50 mg/ml gentamicin, and transferred to 1 ml maturation medium. Control and DRB-treated COC were concurrently placed into maturation
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medium. All cultures were continued for an additional 20 h, with groups of COC from each treatment (Control, DRB) removed every 5 h for assessment of stage of meiosis. This experiment was replicated four times with an average of 20 3 COC per treatment group per time point within each replicate. Oocytes were assessed for stage of meiotic maturation as previously described [12]. Briefly, COC were denuded by vortexing for 90 s, placed on a microscope slide and gently covered with a cover slip on which Vaseline petroleum jelly was placed on two opposing edges. COC were fixed in ethanol–acetic acid (3:1; v/v) for approximately 20 h, stained with 1% orcein in 25% acetic acid, and subsequently de-stained with ethanol–acetic acid. Oocytes were evaluated for stage of meiotic maturation using criteria established by Motlik et al. [29] using differential interference contrast microscopy (magnification, 200). 2.3. Experiment II: developmental competence of COC following DRB arrest The overall design for Experiment II is illustrated in Fig. 1. Both control and treatment media were changed every 4 h throughout the 20 h inhibitor treatment period. All cultures were maintained in an atmosphere of 5% CO2 in air with 100% humidity. At the end of the 20 h inhibitor treatment period, a subset of COC from the DRB-treated group and all COC from the control group were assessed for stage of meiotic maturation as previously described [12].
Experimental Design Control
Treatment Maturation + inhibitor
Inhibitor-free medium 20 h COC culture n = 12
assessment of meiotic stage
n = 15
Wash 5X Control II 20 h COC culture in inhibitor-free maturation media
Fertilization
M-199
mSOF
Development to blastocyst (Day 7, Day 9)
Fertilization
M-199
mSOF
Fig. 1. Experiment II: experimental design. Bovine COC were cultured for 20 h in the presence or absence of the transcriptional inhibitor DRB. A subsample of COC cultured in the presence of DRB and all COC in the control group were then fixed and assessed for meiotic stage. COC in the DRB-treated group were washed, transferred to inhibitor-free medium and initiated maturation with a second group of control COC. After maturation, COC were fertilized and distributed to either TCM-199/serum or mSOF/BSA culture systems. Development was assessed at 72 hpi, Day 7 and Day 9.
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The remaining COC from the DRB treatment group were washed five times in maturation medium and then transferred to inhibitor-free medium for an additional 20 h of culture. This 20 h maturation period was chosen based on the results of Experiment I. A new group of untreated COC were collected, washed and matured simultaneously for use as a control group (see Fig. 1). After maturation, all COC were washed once and placed into 0.75 ml fertilization medium consisting of Tyrode–albumin–lactate–pyruvate (TALP) medium supplemented with 6 mg/ml fatty acid-free BSA and 10 mg/ml heparin [30]. Motile spermatozoa were collected using a swim-up procedure [30] and fertilization was conducted for 18–20 h using a final concentration of 1 106 spermatozoa/ml. Thawed frozen semen from the same sire was used for the production of all embryos. Presumptive zygotes were washed six times in TL-Hepes and transferred, with their cumulus cells, into either 1 ml TCM-199 with 10% ECS and 50 mg/ml gentamicin or into 1 ml modified Synthetic Oviductal Fluid (mSOF) supplemented with 0.6% fatty acid free BSA, 1% (v/v) minimal essential medium non-essential amino acids and 50 mg/ml gentamicin [27]. Cultures in TCM-199-based development medium were performed at 39 8C in an atmosphere of 5% CO2 in air with 100% humidity. Medium was changed at 48 h intervals throughout culture. Cultures in mSOF were performed at 39 8C in an atmosphere of 90%N:5%O2:5%CO2 and the medium was undisturbed throughout the entire development period. At 72 h post insemination (hpi), a subset of zygotes from each treatment group was assessed to determine the percentage of zygotes that cleaved within each experimental replicate. The remaining undisturbed zygotes continued in culture and were assessed for stage of development at 168 hpi (Day 7) and 216 hpi (Day 9). Experiment II was replicated six times with an average of 31 6 COC per treatment group per replicate. 2.4. Statistical analysis In Experiment I, data for the effect of treatment on the percentage of oocytes within specific meiotic stages at different time points from the initiation of treatment were arcsin transformed and analyzed by ANOVA [31]. The statistical model included the effects of replicate, treatment, time and the interaction of treatment by time. Means were separated by Duncan’s Multiple Range Test. In Experiment II, data for the effect of treatment on the percentage of oocytes within specific meiotic stages were arcsin transformed and analyzed using Student’s t tests. Data for the effect of treatment on preimplantation embryo development were arcsin transformed and analyzed by ANOVA using a model that included the main effects of inhibitor (DRB, control), culture system (M199/serum, SOF/BSA) and their interaction [31]. When appropriate, means were separated by Duncan’s Multiple Range Test. Means were considered statistically different at P < 0:05. All data are reported as least squares means S:E. 3. Results 3.1. Experiment I: kinetics of oocyte maturation following DRB arrest Approximately 80% of oocytes in the DRB-treated group were maintained at the GV stage after 20 h of culture. This percentage was lower than the percentage of oocytes in the
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GV oocytes (%)
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*
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*
60 40 20
MI oocytes (%)
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0
5
10
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0
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15
20
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*
*
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Interval (h) Fig. 2. Experiment I: kinetics of oocyte maturation following 20 h of meiotic arrest. COC were cultured for 20 h in DRB-supplemented medium, washed and transferred to DRB-free medium for an additional 20 h of culture. Control (solid line) and DRB-treated (dashed line) COC were placed in DRB-free medium simultaneously and assessed for meiotic stage at 0, 5, 10, 15, and 20 h after the initiation of culture. Panel A: percent GV oocytes; panel B: percent MI oocytes; panel C: percent MII oocytes. P < 0:05, means differ between treatments within time point.
control group found at the GV stage immediately following removal from the follicle (DRB 0 h: 81 3% GV versus Control 0 h: 98 3% GV; P < 0:05; Fig. 2A). After 5 h of culture in DRB-free medium, the majority of the DRB-treated and control oocytes were at the GV stage; however, the percent GV oocytes in the DRB-treated group continued to be lower than control (DRB 5 h: 74 3% GV versus Control 5 h: 98 3% GV; P < 0:05; Fig. 2A). After 10 and 15 h of culture, the majority of oocytes in the DRB-treated group were at
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metaphase I (MI) and this percentage did not differ from their respective control groups (DRB 10 h: 63 5% MI versus Control 10 h: 80 5% MI; DRB 15 h: 60 5% MI versus Control 10 h: 66 5% MI; Fig. 2B). After 20 h of culture, nuclear maturation was completed with the majority of the oocytes in both groups at metaphase II (MII; DRB 20 h: 96 4% MII versus Control 20 h: 99 4% MII; Fig. 2C). 3.2. Experiment II: developmental competence of COC following DRB arrest Consistent with previous reports [12,32,33], bovine COC matured for 20 h in the presence of the transcriptional inhibitor DRB remained at the GV stage (85 3% GV) compared to control COC (1 3% GV; P < 0:05, Fig. 3). After 20 h of culture, the majority of COC in the control group reached metaphase II (96 3% versus 4 3% for control and DRB-treated COC, respectively; P < 0:05, Fig. 3). No significant effect of treatment was found for the percentage of oocytes that remained at MI after 20 h of culture. This observation supported the hypothesis that DRB specifically blocked the progression through GVBD. Significant effects of both DRB treatment and culture system were found on cleavage rates of zygotes when assessed at 72 hpi (DRB: 78 3% versus Control: 90 2%; M199: 89 3% versus mSOF: 80 3%, P < 0:05; Fig. 4). No significant interactions were found between inhibitor treatment and culture system used. The percentage of cleaved zygotes that developed to the blastocyst stage after 168 hpi (Day 7) or 216 hpi (Day 9) was not significantly affected by previous exposure of COC to DRB (Fig. 5A). However, at both Day 7 and Day 9 of culture, a greater proportion of cleaved zygotes developed to the blastocyst stage when cultured in the TCM-199/serum system compared to mSOF/BSA system (Day 7: TCM-199, 23 2% versus mSOF,
100
*
Oocytes (%)
80 60 40 20
* 0
GV
MI
M II
Stage of meiosis Fig. 3. Experiment II: distribution of meiotic stages for bovine COC after 20 h culture in the presence (dark bars) or absence (white bars) of the transcriptional inhibitor DRB. P < 0:05, means differ between treatments within stage of meiosis.
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Cleaved zygotes (%)
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100
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80 60 40 20 0
Cleaved zygotes (%)
Control
100
DRB
(B)
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80 60 40 20 0
M-199
mSOF
Fig. 4. Experiment II: effect of treatment (Control vs. DRB) and culture system (M-199 þ 10% ECS vs. mSOF þ 0:6% BSA) on the proportion of cleaved zygotes by 72 hpi. P < 0:05.
13 2%, P < 0:05; Day 9: TCM-199, 32 3% versus mSOF, 22 3%, P < 0:05, Fig. 5B). There was no interaction was found between DRB treatment and culture system on blastocyst development at either Day 7 or Day 9 of culture.
4. Discussion Results of the present study supported the conclusion that prolonged transcriptional inhibition of oocyte maturation with DRB does not compromise subsequent progression to metaphase II or development to the blastocyst stage. Our results contrast with those of de Wit and Kruip [22] who found that arrest of meiosis by prolonged transcriptional inhibition using a-amanitin was severely detrimental to oocyte developmental competence. In that study, no COC held in prolonged meiotic arrest with a-amanitin developed to the blastocyst stage after fertilization in vitro. The detrimental effect of a-amanitin on the developmental capacity of treated COC may be related to the irreversible nature of the transcriptional block induced by a-amanitin treatment. In the present study, oocytes held in meiotic arrest by a reversible inhibitor of transcription (DRB) resumed meiosis and the proportion of oocytes that reached metaphase II after 20 h of culture in inhibitor-free medium did not differ from control. Furthermore, the kinetics of maturation of DRB-treated COC following transfer to DRB-free medium was comparable to control COC. When expressed as percent of cleaved zygotes, the rate of blastocyst development did not differ between DRB-treated and control COC.
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Blastocysts (%)
(A)
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Control DRB
30 20 10 0
Day 7
Blastocysts (%)
(B)
Day 9 M-199 mSOF
30 20
* *
10 0
Day 7
Day 9
Day of culture Fig. 5. Experiment II: effect of treatment (Control vs. DRB, panel A) and culture system (M-199 þ ECS vs. mSOF þ 0:6% BSA, panel B) on blastocyst development on Day 7 and Day 9 expressed as a percent of cleaved zygotes. P < 0:05 within day.
In Experiment I, approximately 20% of COC in the DRB-treated group were not inhibited from resuming meiosis, and thus underwent GVBD during the initial inhibitor treatment. Perhaps this non-inhibited subset of oocytes did not respond to the FSH priming required for transcriptionally-mediated arrest [12,16] and underwent spontaneous maturation instead. Alternatively, gonadotropin-induced maturation may have already begun in this subset of COC prior to the start of inhibitor treatment and thus the transcriptional inhibitor was no longer effective in preventing GVBD. Independent effects of DRB treatment and type of embryo culture system were found on the percentage of COC that cleaved following fertilization. The most likely explanation for the detrimental effects of DRB exposure on subsequent cleavage rates in Experiment II is related to the observation that approximately 15% of oocytes cultured in the presence of DRB did not become arrested at the GV stage and thus, underwent GVBD. Following an additional 20 h of culture in DRB-free medium, these oocytes probably became aged and were not viable at fertilization, contributing to the reduced rate of oocyte cleavage observed compared to controls. Therefore, data for development to the blastocyst stage were expressed as percent of cleaved zygotes rather than total oocytes. Cleavage percentages reported for each of the culture systems used in the present study were comparable to those reported by other laboratories [34–36]. In the present study, the proportion of COC that cleaved following fertilization was lower for COC cultured in the
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mSOF/BSA culture system compared to the M199/serum system. This observation is in contrast to data that suggests that serum has no beneficial effect on early embryo development [34,36,37]. In the present study, an open-well system was used for the mSOF cultures rather than a microdrop system. The increased culture volume in the mSOF system may have diluted embryotrophic factors secreted by either the cumulus cells or the developing zygotes. In contrast, in the M199/serum culture system, use of the open-well format encouraged development of the cumulus cell monolayer and, therefore, may have enhanced its embryotrophic effects. Development rates to the blastocyst stage were higher in the TCM-199/serum system compared to the mSOF/BSA system, regardless of inhibitor treatment. These results are consistent with the observation that development to the blastocyst stage is significantly increased in association with serum supplementation compared to BSA supplementation [34,36–38]. Serum likely contributes a variety of components that can directly support blastocyst development. In addition, serum may also contribute to a more favorable environment that supports the development of a monolayer of cumulus cells that facilitates development of embryos to the blastocyst stage [39,40]. A greater proportion of oocytes matured in vivo reach the blastocyst stage compared to oocytes matured in vitro [20,41–43]. That in vivo matured COC have greater developmental competencies than in vitro matured COC supports the premise that in vitro maturation systems can be improved. The manipulation of factors that may affect developmental competence while COC are maintained in meiotic arrest needs to be explored [3]. Progesterone treatment, which stimulates transcription of de novo mRNAs through a receptor-mediated mechanism [44], significantly enhanced subsequent oocyte developmental competence when administered during a 6-h period of meiotic arrest maintained by cycloheximide [3]. Similarly, analogs of cAMP have been shown to stimulate progesterone receptor-mediated transcription in the absence of progesterone [44] and increased oocyte developmental competence during a 6-h cycloheximide arrest [45]. Most recently, in porcine COC, induction of LH receptors by exposure to FSH during induced meiotic arrest with IBMX resulted in an increase in developmental competence [46]. Taken together, these observations suggest that mammalian oocytes can respond to the manipulation of factors applied during meiotic arrest that can increase oocyte developmental competence. Based on studies of [3 H]uridine incorporation, the bovine oocyte is transcriptionally active during folliculogenesis [10,47]. Transcriptional activity decreases during antrum formation but a low level of activity is detected in fully-grown mouse [48] and bovine oocytes [10,47]. This low level of transcriptional activity may represent the activation of genes that influence the acquisition of developmental competence as well as the activation of genes that signal the resumption of meiosis. Culture of bovine COC in the presence of DRB would inhibit all transcription initiation, including the generation of mRNAs needed for each of these important physiological functions. Therefore, it is not surprising that an increase in development rates was not observed following DRB treatment in the present study. More importantly, however, subsequent developmental capacity of COC maintained in meiotic arrest by culture in the presence of DRB appeared not to be compromised. Therefore, this DRB-arrest model represents a useful physiological approach for the identification of gene products that regulate the onset of GVBD. Further, identification of
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specific transcripts involved in controlling resumption of meiosis would allow for targeted inhibition of these genes while maintaining continued synthesis of other transcripts important for the acquisition of developmental competence. In summary, bovine COC maintained in meiotic arrest for 20 h by culture in the presence of the transcriptional inhibitor DRB resumed meiosis and maintained their capacity to develop to the blastocyst stage.
Acknowledgements Research supported by USDA Grant 2002-35205-12810 and the North Carolina Agricultural Research Service. The authors acknowledge Drs. Peter W. Farin and Robert M. Petters for critical review of this manuscript. References [1] Blondin P, Coenen K, Guibault L, Sirard M. In vitro competence of bovine embryos: developmental competence is acquired before maturation. Theriogenology 1997;47:1061–75. [2] Hyttel P, Viuff D, Fair T, Laurincik J, Thomsen PD, Callesen H, et al. Ribosomal RNA gene expression and chromosome aberrations in bovine oocytes and preimplantation embryos. Reproduction 2001;122:21–30. [3] Sirard MA. Resumption of meiosis: mechanism involved in meiotic progression and its relation with developmental competence. Theriogenology 2001;55:1241–54. [4] Fair T, Hulshof SC, Hyttel P, Greve T, Boland M. Oocyte ultrastructure in bovine primordial to early tertiary follicles. Anat Embryol (Berl) 1997;195:327–36. [5] de Matos DG, Furnus CC, Moses DF, Baldassarre H. Effect of cysteamine on glutathione level and developmental capacity of bovine oocyte matured in vitro. Mol Reprod Dev 1995;42:432–6. [6] Furnus CC, de Matos DG, Moses DF. Cumulus expansion during in vitro maturation of bovine oocytes: relationship with intracellular glutathione level and its role on subsequent embryo development. Mol Reprod Dev 1998;51:76–83. [7] Brackett BG. In vitro oocyte maturation and fertilization. J Anim Sci 1985;61:14–24. [8] Fulka Jr J, First NL, Moor RM. Nuclear and cytoplasmic determinants involved in the regulation of mammalian oocyte maturation. Mol Hum Reprod 1998;4:41–9. [9] Tsafriri A, Lieberman ME, Koch Y, Bauminger S, Chobsieng P, Zor U, et al. Capacity of immunologically purified FSH to stimulate cyclic AMP accumulation and steroidogenesis in Graafian follicles and to induce ovum maturation and ovulation in the rat. Endocrinology 1976;98:655–61. [10] Fair T, Hyttel P, Greve T. Bovine oocyte diameter in relation to maturational competence and transcriptional activity. Mol Reprod Dev 1995;42:437–42. [11] Arlotto T, Schwartz JL, First NL, Leibfried-Rutledge ML. Aspects of follicle and oocyte stage that affect in vitro maturation and development of bovine oocytes. Theriogenology 1996;45:943–56. [12] Farin CE, Yang L. Inhibition of germinal vesicle breakdown in bovine oocytes by 5,6-dichloro-1-b-Dribofuranosylbenzimidazole (DRB). Mol Reprod Dev 1994;37:284–92. [13] Meinecke B, Meinecke-Tillmann S. Effects of a-amanitin on nuclear maturation of porcine oocytes in vitro. J Reprod Fertil 1993;98:195–201. [14] Osborn JC, Moor RM. Time-dependent effects of a-amanitin on nuclear maturation and protein synthesis in mammalian oocytes. J Embryol Exp Morphol 1983;73:317–38. [15] Kastrop PM, Hulshof SC, Bevers MM, Destree OH, Kruip TA. The effects of a-amanitin and cycloheximide on nuclear progression, protein synthesis, and phosphorylation during bovine oocyte maturation in vitro. Mol Reprod Dev 1991;28:249–54. [16] Rodriguez KF, Petters RM, Crosier AE, Farin CE. Roles of gene transcription and PKA subtype activation in maturation of murine oocytes. Reproduction 2002;123:799–806.
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[17] Wiemer KE, Watson AJ, Polanski V, McKenna AI, Fick GH, Schultz GA. Effects of maturation and coculture treatments on the developmental capacity of early bovine embryos. Mol Reprod Dev 1991;30:330–8. [18] Izadyar F, Colenbrander B, Bevers MM. In vitro maturation of bovine oocytes in the presence of growth hormone accelerates nuclear maturation and promotes subsequent embryonic development. Mol Reprod Dev 1996;45:372–7. [19] Ward F, Enright B, Rizos D, Boland M, Lonergan P. Optimization of in vitro bovine embryo production: effect of duration of maturation, length of gamete co-incubation, sperm concentration and sire. Theriogenology 2002;57:2105–17. [20] Rizos D, Ward F, Duffy P, Boland MP, Lonergan P. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol Reprod Dev 2002;61:234–48. [21] Mermillod P, Tomanek M, Marchal R, Meijer L. High developmental competence of cattle oocytes maintained at the germinal vesicle stage for 24 h in culture by specific inhibition of MPF kinase activity. Mol Reprod Dev 2000;55:89–95. [22] de Wit AA, Kruip TA. Bovine cumulus-oocyte-complex-quality is reflected in sensitivity for alphaamanitin, oocyte-diameter and developmental capacity. Anim Reprod Sci 2001;65:51–65. [23] Zubay G. Biochemistry. New York: Addison-Wesley Publishing Co.; 1983. [24] Zandomeni R, Bunick D, Ackerman S, Mittleman B, Weinmann R. Mechanism of action of DRB. III. Effect on specific in vitro initiation of transcription. J Mol Biol 1983;167:561–74. [25] Lonergan P, Khatir H, Carolan C, Mermillod P. Bovine blastocyst production in vitro after inhibition of oocyte meiotic resumption for 24 h. J Reprod Fertil 1997;109:355–65. [26] Krisher RL, Lane M, Bavister BD. Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media. Biol Reprod 1999;60:1345–52. [27] Crosier AE, Farin PW, Dykstra MJ, Alexander JE, Farin CE. Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro. Biol Reprod 2001;64:1375–85. [28] Farin PW, Crosier AE, Farin CE. Influence of in vitro systems on embryo survival and fetal development in cattle. Theriogenology 2001;55:151–70. [29] Motlik J, Koefoed-Johnsen HH, Fulka J. Breakdown of the germinal vesicle in bovine oocytes cultivated in vitro. J Exp Zool 1978;205:377–83. [30] Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH, First NL. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 1986;25:591–600. [31] SAS. SAS User’s guide: statistics, release 6.03 ed. Cary, NC: Statistical Analysis System Institute; 1988. [32] Martus NS, Farin CE. Effectiveness of DRB for inhibiting germinal vesicle breakdown in bovine oocytes. Theriogenology 1994;42:1295–302. [33] Wolf CJ, Farin CE. Effect of gonadotropins on the ability of 5,6-dichloro-1-b-D-ribofuranosylbenzamidazole (DRB) to inhibit germinal vesicle breakdown in bovine oocytes. Theriogenology 1996;46:760–8. [34] Wang S, Liu Y, Holyoak GR, Bunch TD. The effects of bovine serum albumin and fetal bovine serum on the development of pre- and postcleavage-stage bovine embryos cultured in modified CR2 and M199 media. Anim Reprod Sci 1997;48:37–45. [35] Gutierrez-Adan A, Lonergan P, Rizos D, Ward FA, Boland MP, Pintado B, et al. Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos. Theriogenology 2001;55:1117–26. [36] Rizos D, Gutierrez-Adan A, Perez-Garnelo S, De La Fuente J, Boland MP, Lonergan P. Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol Reprod 2003;68:236–43. [37] Pinyopummintr T, Bavister BD. In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined, protein-free culture media. Biol Reprod 1991;45:736–42. [38] Wrenzycki C, Herrmann D, Keskintepe L, Martins Jr A, Sirisathien S, Brackett B, et al. Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum Reprod 2001;16:893–901. [39] Thomas WK, Seidel Jr GE. Effects of cumulus cells on culture of bovine embryos derived from oocytes matured and fertilized in vitro. J Anim Sci 1993;71:2506–10. [40] Zhang L, Jiang S, Wozniak PJ, Yang X, Godke RA. Cumulus cell function during bovine oocyte maturation, fertilization, and embryo development in vitro. Mol Reprod Dev 1995;40:338–44.
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[41] Dieleman SJ, Hendriksen PJ, Viuff D, Thomsen PD, Hyttel P, Knijn HM, et al. Effects of in vivo prematuration and in vivo final maturation on developmental capacity and quality of pre-implantation embryos. Theriogenology 2002;57:5–20. [42] Hendriksen PJ, Vos PL, Steenweg WN, Bevers MM, Dieleman SJ. Bovine follicular development and its effect on the in vitro competence of oocytes. Theriogenology 2000;53:11–20. [43] van de Leemput EE, Vos PL, Zeinstra EC, Bevers MM, van der Weijden GC, Dieleman SJ. Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory LH surge. Theriogenology 1999;52:335–49. [44] Mahesh VB, Brann DW, Hendry LB. Diverse modes of action of progesterone and its metabolites. J Steroid Biochem Mol Biol 1996;56:209–19. [45] Guixue Z, Luciano AM, Coenen K, Gandolfi F, Sirard MA. The influence of cAMP before or during bovine oocyte maturation on embryonic developmental competence. Theriogenology 2001;55:1733–43. [46] Shimada M, Nishibori M, Isobe N, Kawano N, Terada T. Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes. Biol Reprod 2003;68:1142–9. [47] Memili E, First NL. Developmental changes in RNA polymerase II in bovine oocytes, early embryos, and effect of alpha-amanitin on embryo development. Mol Reprod Dev 1998;51:381–9. [48] Wassarman PM, Letourneau GE. RNA synthesis in fully-grown mouse oocytes. Nature 1976;261:73–4.
Developmental capacity of bovine cumulus oocyte complexes after transcriptional inhibition of germinal vesicle breakdown K.F. Rodriguez, C.E. Farin* Department of Animal Science, North Carolina State University, Box 7621, 231 B Polk Hall, Raleigh, NC 27695-7621, USA Received 12 November 2002; accepted 28 August 2003
Abstract Oocytes cultured in the presence of FSH and the transcriptional inhibitor, 5,6-dichloro-1-b-Dribofuranosylbenzimidazole (DRB), remain in meiotic arrest at the germinal vesicle (GV) stage. The objectives of this study were to assess the kinetics of maturation and the developmental capacity of bovine cumulus oocyte complexes (COC) following release from prolonged meiotic arrest by DRB. In Experiment I, COC were cultured for 20 h in Tissue culture medium (TCM)-199 supplemented with 10% estrus cow serum (ECS), 5 mg/ml FSH and 1 mg/ml estradiol in the presence of 120 mM DRB. COC were then released from meiotic arrest and cultured for 20 h in DRB-free medium. Control COC were cultured for 20 h in DRB-free medium, with culture initiated concomitant to the release of DRB-treated COC from meiotic arrest. Nuclear maturation was assessed after 0, 5, 10, 15, and 20 h of culture in DRB-free medium. The proportion of DRB-arrested oocytes reaching metaphase II (MII) following 20 h culture in DRB-free medium was not significantly different from controls (96 4% versus 99 4%). In Experiment II, COC were cultured for 20 h in TCM-199 supplemented with 10% ECS, 10 mg/ml LH, 5 mg/ml FSH, and 1 mg/ml estradiol in the presence or absence of 120 mM DRB. COC in the DRB-treated group were then washed and matured coincident with a second group of control COC for 20 h in DRB-free medium. COC in both groups were fertilized and then randomly assigned to one of two culture systems: TCM-199 þ 10%ECS or mSOF þ 0:6% fatty acid-free BSA. Development was assessed at 72 h post insemination (hpi), 168 hpi (Day 7) and 216 hpi (Day 9). In this experiment, culture with DRB-arrested oocyte maturation at the GV stage (DRB, 85 3% GV; Control, 2 3% GV; P < 0:001). Following release from arrest, maturation and fertilization, the proportion of COC that cleaved by 72 hpi was decreased by treatment with DRB (DRB: 78 3% versus Control: 90 3%; P < 0:05). However, no effect of DRB was found on the proportion of cleaved zygotes that reached the blastocyst stage on either Day 7 or Day 9 of culture (Day 7: DRB 16 2% versus Control, 21 2%; Day 9: DRB 23 3% versus Control, 31 3%). More embryos reached the blastocyst stage in the * Corresponding author. Tel.: þ1-919-515-4022; fax: þ1-919-515-7780. E-mail address: [email protected] (C.E. Farin).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.08.016
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TCM-199/serum culture system compared to the mSOF/BSA system on both Days 7 and 9 (Day 7: TCM-199, 23 2% versus mSOF, 13 2%, P < 0:05; Day 9: TCM-199, 32 3% versus mSOF, 22 3%, P < 0:05). In summary, bovine COC maintained in meiotic arrest for 20 h by culture in the presence of the transcriptional inhibitor DRB retained their capacity to develop to the blastocyst stage after fertilization in vitro. # 2003 Elsevier Inc. All rights reserved. Keywords: Oocyte; Meiosis; Transcription; Blastocyst; Bovine
1. Introduction Throughout follicular development, oocytes remain arrested at prophase I of meiosis. During this period of meiotic arrest, oocyte growth continues and maternal mRNA transcripts required for acquisition of developmental competence accumulate in the cytoplasm [1–3]. Both cytoplasmic and nuclear maturation must occur for the oocyte to become competent to support subsequent post-fertilization development. Cytoplasmic maturation includes not only the accumulation of maternal mRNA, but also cellular changes such as alignment of cortical granules [4], increased lipid accumulation [4] and accumulation of glutathione [5,6]. Nuclear maturation refers to acquisition of the ability to undergo dissolution of the germinal vesicle (nuclear membrane), condensation of the chromosomes, release of the first polar body, and subsequent arrest at metaphase II (MII) [7,8]. In vivo, resumption of meiosis is induced by the preovulatory surge of gonadotropins [9]. However, when bovine cumulus oocyte complexes (COC) are aspirated from antral follicles greater than 2 mm in diameter and cultured in vitro, they resume meiosis either spontaneously or in the presence of gonadotropins [10,11]. De novo transcription is required for gonadotropin-mediated, but not spontaneous, oocyte maturation [12]. Specific inhibitors of transcription, such as a-amanitin or 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB), have been used to arrest gonadotropin-mediated resumption of meiosis in the pig [13], sheep [14], cow [12,15], and mouse [16]. For transcriptional inhibitors to effectively arrest maturation at the germinal vesicle (GV) stage, several layers of cumulus cells must surround the oocytes and gonadotropins must be present in the maturation medium [12,16]. These observations support the conclusion that gonadotropin stimulation results in a transcriptional event that occurs in the cumulus cells that is required for initiation of GVBD [12,16]. Only 30–40% of bovine oocytes that undergo maturation and fertilization in vitro develop to the blastocyst stage [17–19]; in contrast, approximately 80% of oocytes matured and fertilized in vivo reach the blastocyst stage [20]. It has been suggested that the developmental competence of in vitro matured oocytes might be increased if oocytes could be maintained in meiotic arrest allowing an additional opportunity for cytoplasmic maturation to occur [3,21]. Roscovitine, a purine that specifically inhibits maturation promoting factor (MPF) activity and blocks cell cycle progression, has been used to maintain bovine oocytes at the GV stage without compromising their subsequent developmental potential [21]. In contrast, developmental capacity after fertilization of bovine COC maintained in transcriptional arrest of meiosis with a-amanitin is severely reduced [22]; this transcriptional inhibitor specifically blocks nucleoplasmic RNA synthesis by
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irreversibly binding to RNA polymerase II [23]. That the developmental capacity following a-amanitin exposure may be compromised is consistent with its mechanism of action. Alternatively, DRB specifically and reversibly blocks transcription by preventing the formation of a stable transcription initiation complex [24]. Because the action of DRB is highly reversible [24], it would be anticipated that oocyte developmental competence may be better maintained following meiotic arrest with this inhibitor. There were differences in the proportion of COC that reached the blastocyst stage, depending on the culture system utilized and specifically on the presence or absence of serum in the culture system [20,25,26]. Furthermore, in vitro culture systems can influence gene expression, morphology and blastocyst development [26–28]. Therefore, the developmental capacity of oocytes maintained in meiotic arrest by prolonged culture in the presence of a transcriptional inhibitor may also be influenced by culture conditions following fertilization. The objectives of this study were first, to analyze the kinetics of oocyte maturation following release from transcriptional inhibition of meiosis by DRB; and second, to assess the developmental capacity of bovine COC that were maintained in prolonged meiotic arrest with DRB using two different culture systems.
2. Materials and methods 2.1. Reagents and media Tissue culture medium (TCM-199 with Earl’s salts) was purchased from Gibco BRL (Grand Island, NY, USA). Equine pituitary LH (11.5 NIH LH-S1 U/mg), porcine pituitary FSH (50 mg/vial Armour FSH standard) and the transcriptional inhibitor DRB were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents and medium supplements were of tissue culture grade and purchased from Sigma Chemical Co. 2.2. Experiment I: kinetics of oocyte maturation following DRB arrest Ovaries were collected at a local abattoir and transported to the laboratory at room temperature in saline with 0.75 mg/ml penicillin. Approximately 1.5 h after ovary collection, follicles 2–8 mm in diameter were aspirated using an 18-gauge needle and 10 ml syringe. COC with several layers of cumulus cells were collected, washed five times in modified Tyrode’s medium (TL-Hepes), and cultured for 20 h in 1 ml of TCM-199, supplemented with 10% heat-inactivated estrus cow serum (ECS), 5 mg/ml FSH, 1 mg/ml estradiol, 200 mM pyruvate, and 50 mg/ml gentamicin in the presence of either DRB (120 mM in 0.2% DMSO) or vehicle control (0.2% DMSO). All maturation cultures were maintained at 39 8C in an atmosphere of 5% CO2 in air with 100% humidity. COC were cultured in the presence of DRB for a total of 20 h. The treatment medium was changed every 4 h throughout this period to maximally inhibit oocyte maturation [12]. At the end of the treatment period, COC were washed six times in maturation medium (TCM-199) supplemented with 10% heat-inactivated ECS, 10 mg/ml LH, 5 mg/ml FSH, 1 mg/ml estradiol, 200 mM pyruvate, and 50 mg/ml gentamicin, and transferred to 1 ml maturation medium. Control and DRB-treated COC were concurrently placed into maturation
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medium. All cultures were continued for an additional 20 h, with groups of COC from each treatment (Control, DRB) removed every 5 h for assessment of stage of meiosis. This experiment was replicated four times with an average of 20 3 COC per treatment group per time point within each replicate. Oocytes were assessed for stage of meiotic maturation as previously described [12]. Briefly, COC were denuded by vortexing for 90 s, placed on a microscope slide and gently covered with a cover slip on which Vaseline petroleum jelly was placed on two opposing edges. COC were fixed in ethanol–acetic acid (3:1; v/v) for approximately 20 h, stained with 1% orcein in 25% acetic acid, and subsequently de-stained with ethanol–acetic acid. Oocytes were evaluated for stage of meiotic maturation using criteria established by Motlik et al. [29] using differential interference contrast microscopy (magnification, 200). 2.3. Experiment II: developmental competence of COC following DRB arrest The overall design for Experiment II is illustrated in Fig. 1. Both control and treatment media were changed every 4 h throughout the 20 h inhibitor treatment period. All cultures were maintained in an atmosphere of 5% CO2 in air with 100% humidity. At the end of the 20 h inhibitor treatment period, a subset of COC from the DRB-treated group and all COC from the control group were assessed for stage of meiotic maturation as previously described [12].
Experimental Design Control
Treatment Maturation + inhibitor
Inhibitor-free medium 20 h COC culture n = 12
assessment of meiotic stage
n = 15
Wash 5X Control II 20 h COC culture in inhibitor-free maturation media
Fertilization
M-199
mSOF
Development to blastocyst (Day 7, Day 9)
Fertilization
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Fig. 1. Experiment II: experimental design. Bovine COC were cultured for 20 h in the presence or absence of the transcriptional inhibitor DRB. A subsample of COC cultured in the presence of DRB and all COC in the control group were then fixed and assessed for meiotic stage. COC in the DRB-treated group were washed, transferred to inhibitor-free medium and initiated maturation with a second group of control COC. After maturation, COC were fertilized and distributed to either TCM-199/serum or mSOF/BSA culture systems. Development was assessed at 72 hpi, Day 7 and Day 9.
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The remaining COC from the DRB treatment group were washed five times in maturation medium and then transferred to inhibitor-free medium for an additional 20 h of culture. This 20 h maturation period was chosen based on the results of Experiment I. A new group of untreated COC were collected, washed and matured simultaneously for use as a control group (see Fig. 1). After maturation, all COC were washed once and placed into 0.75 ml fertilization medium consisting of Tyrode–albumin–lactate–pyruvate (TALP) medium supplemented with 6 mg/ml fatty acid-free BSA and 10 mg/ml heparin [30]. Motile spermatozoa were collected using a swim-up procedure [30] and fertilization was conducted for 18–20 h using a final concentration of 1 106 spermatozoa/ml. Thawed frozen semen from the same sire was used for the production of all embryos. Presumptive zygotes were washed six times in TL-Hepes and transferred, with their cumulus cells, into either 1 ml TCM-199 with 10% ECS and 50 mg/ml gentamicin or into 1 ml modified Synthetic Oviductal Fluid (mSOF) supplemented with 0.6% fatty acid free BSA, 1% (v/v) minimal essential medium non-essential amino acids and 50 mg/ml gentamicin [27]. Cultures in TCM-199-based development medium were performed at 39 8C in an atmosphere of 5% CO2 in air with 100% humidity. Medium was changed at 48 h intervals throughout culture. Cultures in mSOF were performed at 39 8C in an atmosphere of 90%N:5%O2:5%CO2 and the medium was undisturbed throughout the entire development period. At 72 h post insemination (hpi), a subset of zygotes from each treatment group was assessed to determine the percentage of zygotes that cleaved within each experimental replicate. The remaining undisturbed zygotes continued in culture and were assessed for stage of development at 168 hpi (Day 7) and 216 hpi (Day 9). Experiment II was replicated six times with an average of 31 6 COC per treatment group per replicate. 2.4. Statistical analysis In Experiment I, data for the effect of treatment on the percentage of oocytes within specific meiotic stages at different time points from the initiation of treatment were arcsin transformed and analyzed by ANOVA [31]. The statistical model included the effects of replicate, treatment, time and the interaction of treatment by time. Means were separated by Duncan’s Multiple Range Test. In Experiment II, data for the effect of treatment on the percentage of oocytes within specific meiotic stages were arcsin transformed and analyzed using Student’s t tests. Data for the effect of treatment on preimplantation embryo development were arcsin transformed and analyzed by ANOVA using a model that included the main effects of inhibitor (DRB, control), culture system (M199/serum, SOF/BSA) and their interaction [31]. When appropriate, means were separated by Duncan’s Multiple Range Test. Means were considered statistically different at P < 0:05. All data are reported as least squares means S:E. 3. Results 3.1. Experiment I: kinetics of oocyte maturation following DRB arrest Approximately 80% of oocytes in the DRB-treated group were maintained at the GV stage after 20 h of culture. This percentage was lower than the percentage of oocytes in the
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GV oocytes (%)
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*
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*
*
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Interval (h) Fig. 2. Experiment I: kinetics of oocyte maturation following 20 h of meiotic arrest. COC were cultured for 20 h in DRB-supplemented medium, washed and transferred to DRB-free medium for an additional 20 h of culture. Control (solid line) and DRB-treated (dashed line) COC were placed in DRB-free medium simultaneously and assessed for meiotic stage at 0, 5, 10, 15, and 20 h after the initiation of culture. Panel A: percent GV oocytes; panel B: percent MI oocytes; panel C: percent MII oocytes. P < 0:05, means differ between treatments within time point.
control group found at the GV stage immediately following removal from the follicle (DRB 0 h: 81 3% GV versus Control 0 h: 98 3% GV; P < 0:05; Fig. 2A). After 5 h of culture in DRB-free medium, the majority of the DRB-treated and control oocytes were at the GV stage; however, the percent GV oocytes in the DRB-treated group continued to be lower than control (DRB 5 h: 74 3% GV versus Control 5 h: 98 3% GV; P < 0:05; Fig. 2A). After 10 and 15 h of culture, the majority of oocytes in the DRB-treated group were at
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metaphase I (MI) and this percentage did not differ from their respective control groups (DRB 10 h: 63 5% MI versus Control 10 h: 80 5% MI; DRB 15 h: 60 5% MI versus Control 10 h: 66 5% MI; Fig. 2B). After 20 h of culture, nuclear maturation was completed with the majority of the oocytes in both groups at metaphase II (MII; DRB 20 h: 96 4% MII versus Control 20 h: 99 4% MII; Fig. 2C). 3.2. Experiment II: developmental competence of COC following DRB arrest Consistent with previous reports [12,32,33], bovine COC matured for 20 h in the presence of the transcriptional inhibitor DRB remained at the GV stage (85 3% GV) compared to control COC (1 3% GV; P < 0:05, Fig. 3). After 20 h of culture, the majority of COC in the control group reached metaphase II (96 3% versus 4 3% for control and DRB-treated COC, respectively; P < 0:05, Fig. 3). No significant effect of treatment was found for the percentage of oocytes that remained at MI after 20 h of culture. This observation supported the hypothesis that DRB specifically blocked the progression through GVBD. Significant effects of both DRB treatment and culture system were found on cleavage rates of zygotes when assessed at 72 hpi (DRB: 78 3% versus Control: 90 2%; M199: 89 3% versus mSOF: 80 3%, P < 0:05; Fig. 4). No significant interactions were found between inhibitor treatment and culture system used. The percentage of cleaved zygotes that developed to the blastocyst stage after 168 hpi (Day 7) or 216 hpi (Day 9) was not significantly affected by previous exposure of COC to DRB (Fig. 5A). However, at both Day 7 and Day 9 of culture, a greater proportion of cleaved zygotes developed to the blastocyst stage when cultured in the TCM-199/serum system compared to mSOF/BSA system (Day 7: TCM-199, 23 2% versus mSOF,
100
*
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80 60 40 20
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GV
MI
M II
Stage of meiosis Fig. 3. Experiment II: distribution of meiotic stages for bovine COC after 20 h culture in the presence (dark bars) or absence (white bars) of the transcriptional inhibitor DRB. P < 0:05, means differ between treatments within stage of meiosis.
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Cleaved zygotes (%)
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Fig. 4. Experiment II: effect of treatment (Control vs. DRB) and culture system (M-199 þ 10% ECS vs. mSOF þ 0:6% BSA) on the proportion of cleaved zygotes by 72 hpi. P < 0:05.
13 2%, P < 0:05; Day 9: TCM-199, 32 3% versus mSOF, 22 3%, P < 0:05, Fig. 5B). There was no interaction was found between DRB treatment and culture system on blastocyst development at either Day 7 or Day 9 of culture.
4. Discussion Results of the present study supported the conclusion that prolonged transcriptional inhibition of oocyte maturation with DRB does not compromise subsequent progression to metaphase II or development to the blastocyst stage. Our results contrast with those of de Wit and Kruip [22] who found that arrest of meiosis by prolonged transcriptional inhibition using a-amanitin was severely detrimental to oocyte developmental competence. In that study, no COC held in prolonged meiotic arrest with a-amanitin developed to the blastocyst stage after fertilization in vitro. The detrimental effect of a-amanitin on the developmental capacity of treated COC may be related to the irreversible nature of the transcriptional block induced by a-amanitin treatment. In the present study, oocytes held in meiotic arrest by a reversible inhibitor of transcription (DRB) resumed meiosis and the proportion of oocytes that reached metaphase II after 20 h of culture in inhibitor-free medium did not differ from control. Furthermore, the kinetics of maturation of DRB-treated COC following transfer to DRB-free medium was comparable to control COC. When expressed as percent of cleaved zygotes, the rate of blastocyst development did not differ between DRB-treated and control COC.
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Blastocysts (%)
(A)
1507
Control DRB
30 20 10 0
Day 7
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(B)
Day 9 M-199 mSOF
30 20
* *
10 0
Day 7
Day 9
Day of culture Fig. 5. Experiment II: effect of treatment (Control vs. DRB, panel A) and culture system (M-199 þ ECS vs. mSOF þ 0:6% BSA, panel B) on blastocyst development on Day 7 and Day 9 expressed as a percent of cleaved zygotes. P < 0:05 within day.
In Experiment I, approximately 20% of COC in the DRB-treated group were not inhibited from resuming meiosis, and thus underwent GVBD during the initial inhibitor treatment. Perhaps this non-inhibited subset of oocytes did not respond to the FSH priming required for transcriptionally-mediated arrest [12,16] and underwent spontaneous maturation instead. Alternatively, gonadotropin-induced maturation may have already begun in this subset of COC prior to the start of inhibitor treatment and thus the transcriptional inhibitor was no longer effective in preventing GVBD. Independent effects of DRB treatment and type of embryo culture system were found on the percentage of COC that cleaved following fertilization. The most likely explanation for the detrimental effects of DRB exposure on subsequent cleavage rates in Experiment II is related to the observation that approximately 15% of oocytes cultured in the presence of DRB did not become arrested at the GV stage and thus, underwent GVBD. Following an additional 20 h of culture in DRB-free medium, these oocytes probably became aged and were not viable at fertilization, contributing to the reduced rate of oocyte cleavage observed compared to controls. Therefore, data for development to the blastocyst stage were expressed as percent of cleaved zygotes rather than total oocytes. Cleavage percentages reported for each of the culture systems used in the present study were comparable to those reported by other laboratories [34–36]. In the present study, the proportion of COC that cleaved following fertilization was lower for COC cultured in the
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mSOF/BSA culture system compared to the M199/serum system. This observation is in contrast to data that suggests that serum has no beneficial effect on early embryo development [34,36,37]. In the present study, an open-well system was used for the mSOF cultures rather than a microdrop system. The increased culture volume in the mSOF system may have diluted embryotrophic factors secreted by either the cumulus cells or the developing zygotes. In contrast, in the M199/serum culture system, use of the open-well format encouraged development of the cumulus cell monolayer and, therefore, may have enhanced its embryotrophic effects. Development rates to the blastocyst stage were higher in the TCM-199/serum system compared to the mSOF/BSA system, regardless of inhibitor treatment. These results are consistent with the observation that development to the blastocyst stage is significantly increased in association with serum supplementation compared to BSA supplementation [34,36–38]. Serum likely contributes a variety of components that can directly support blastocyst development. In addition, serum may also contribute to a more favorable environment that supports the development of a monolayer of cumulus cells that facilitates development of embryos to the blastocyst stage [39,40]. A greater proportion of oocytes matured in vivo reach the blastocyst stage compared to oocytes matured in vitro [20,41–43]. That in vivo matured COC have greater developmental competencies than in vitro matured COC supports the premise that in vitro maturation systems can be improved. The manipulation of factors that may affect developmental competence while COC are maintained in meiotic arrest needs to be explored [3]. Progesterone treatment, which stimulates transcription of de novo mRNAs through a receptor-mediated mechanism [44], significantly enhanced subsequent oocyte developmental competence when administered during a 6-h period of meiotic arrest maintained by cycloheximide [3]. Similarly, analogs of cAMP have been shown to stimulate progesterone receptor-mediated transcription in the absence of progesterone [44] and increased oocyte developmental competence during a 6-h cycloheximide arrest [45]. Most recently, in porcine COC, induction of LH receptors by exposure to FSH during induced meiotic arrest with IBMX resulted in an increase in developmental competence [46]. Taken together, these observations suggest that mammalian oocytes can respond to the manipulation of factors applied during meiotic arrest that can increase oocyte developmental competence. Based on studies of [3 H]uridine incorporation, the bovine oocyte is transcriptionally active during folliculogenesis [10,47]. Transcriptional activity decreases during antrum formation but a low level of activity is detected in fully-grown mouse [48] and bovine oocytes [10,47]. This low level of transcriptional activity may represent the activation of genes that influence the acquisition of developmental competence as well as the activation of genes that signal the resumption of meiosis. Culture of bovine COC in the presence of DRB would inhibit all transcription initiation, including the generation of mRNAs needed for each of these important physiological functions. Therefore, it is not surprising that an increase in development rates was not observed following DRB treatment in the present study. More importantly, however, subsequent developmental capacity of COC maintained in meiotic arrest by culture in the presence of DRB appeared not to be compromised. Therefore, this DRB-arrest model represents a useful physiological approach for the identification of gene products that regulate the onset of GVBD. Further, identification of
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specific transcripts involved in controlling resumption of meiosis would allow for targeted inhibition of these genes while maintaining continued synthesis of other transcripts important for the acquisition of developmental competence. In summary, bovine COC maintained in meiotic arrest for 20 h by culture in the presence of the transcriptional inhibitor DRB resumed meiosis and maintained their capacity to develop to the blastocyst stage.
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