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Gamete Biology |
Follicle Biology Laboratory, Vrije Universiteit Brussel, Brussels, 1090 Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, fertilization, meiosis, oocyte development, phosphodiesterases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These oocytes, though, are not necessarily competent to support normal fertilization and further embryonic development. The poor embryo developmental rates are presumably due to a suboptimal oocyte development in vitro due to an asynchronous nuclear and cytoplasmic maturation [3]. In vivo, the inhibitory influences on nuclear maturation provide an optimal balance between nuclear and cytoplasmic maturation processes. The overall lower developmental capacity of oocytes matured in vitro is inherent to the heterogeneity of the starting population. GV-stage oocytes isolated from follicles stimulated by exogenous gonadotropin are heterogeneous in size, number of cumulus cells, and oocyte cytoplasm microtubule organization [4, 5]. GV oocytes from antral follicles of adult rodents [6–8] and primates [9, 10] also present heterogeneity in chromatin configuration. After Hoechst staining, surrounded nucleolus (SN) or nonsurrounded nucleolus (NSN) can be observed. These phenotypical observations are correlated to cellular activity. The transition from NSN to SN configuration in the oocytes has been related to transition from active to inactive transcription [8, 11, 12]. Repression of transcriptional activity is a characteristic of oocytes in preovulatory follicles [5, 8, 12] and has also been correlated to the achievement of oocyte developmental competence [6, 7].
It has been hypothesized that an in vitro prematuration period of oocytes retrieved from small antral follicles might lead to an improved oocyte cytoplasmic maturation [13]. A prematuration period in vitro could very well contribute to synchronizing the population of immature oocytes, resulting in an overall improved outcome. A prematuration culture can only be achieved by blocking the inevitable spontaneous nuclear maturation that starts immediately after the oocyte has been disconnected from the somatic compartment of the follicle. The arrest of meiosis can effectively be induced pharmacologically by manipulating the intraoocyte cAMP levels [14].
Basal physiological levels of cAMP are maintained by synthesis, via adenylate cyclase, and degradation, via the cyclic nucleotide phosphodiesterases (PDEs), a group of enzymes that catalyze the hydrolysis of 3', 5'-cyclic nucleotide to inactive 5'-nucleotide metabolites by cleaving the phosphodiester bond of the cyclic phosphate ring. This large group of proteins consists of at least 11 gene families (types) identified in mammals [15], and some PDE families consist of several subtypes and numerous PDE isoform-splice variants.
PDE3A mRNA, which was initially named PDE3B, is expressed in rodents [16, 17] and in human oocytes [18]. Selective PDE inhibitors were used and have emphasized the importance of a PDE3A enzyme in the regulation of oocyte meiosis [16, 19, 20]. PDE3 inhibitors have been demonstrated to act directly in the oocyte, whereas PDE4 is mainly involved in the metabolization of cAMP in granulosa cells [16]. PDE3 inhibitors have been shown to successfully suppress oocyte nuclear maturation without affecting follicle rupture after gonadotropin stimulation in rats [19]. However, no studies have aimed to investigate the developmental competence of oocytes after reversal from in vitro-induced meiosis arrest by any PDE3 inhibitor. In this study, we tested the hypothesis that making use of a PDE3 inhibitor, Org 9935, to arrest GV-stage oocytes from immature antral mouse follicles for 24 h could benefit developmental competence after in vitro maturation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice used for retrieval of oocytes were 8-wk-old F1 hybrid (C57Bl/6J x CBA/ca), housed and bred according to national legislation for animal care and with the consent of the ethical committee (project 01-395-1).
Collection of Oocytes
Mice were primed with intraperitoneal injection of 5 IU eCG (Folligon; Intervet, Mechelen, Belgium). Females were killed 24 or 48 h later by cervical dislocation. Ovaries were collected in 2 ml of washing medium consisting of L15 Leibovitz-glutamax supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 µM PDE3 inhibitor (Org9935; a gift from Organon, Oss, The Netherlands). Cumulus-oocyte complexes (COCs) were obtained from ovaries by puncturing antral follicles with sterile needles in washing medium. Only immature oocytes surrounded by a compact cumulus-cell mass were collected.
As in vivo controls, ovulated oocytes were obtained by priming mice with 5 IU/ml eCG followed by 5 IU/ml hCG (Chorulon, Intervet) 24 or 48 h later. Ovulated COC were recovered from the ampullae 18 h after hCG priming.
Culture of Oocytes
In vitro maturation (IVM) medium consisted of 
-MEM with glutamax (Life Technologies, Merelbeke, Belgium) supplemented with 5% FCS, 10 mIU/ml recombinant follicle stimulating hormone (rFSH; Ares Serono International, Geneva, Switzerland), 5 ng/ml insulin, 5 µg/ml transferrin, and 5 ng/ml sodium selenite (Sigma, Bornem, Belgium).
IVM arrest was obtained with PDE3 inhibitor, Org 9935, dissolved in dimethylsulfoxide (DMSO), added at a final concentration of 10 µM to the IVM culture medium (final concentration of DMSO was 0.1%). The impact of 0.1% DMSO on oocytes was verified in a preliminary experiment applying it during 18-h IVM, and was revealed not to affect fertilization and embryo developmental capacity of COC after in vitro fertilization (IVF; data not shown).
Removal of PDE3 inhibitor was performed by washing the COC in three steps in inhibitor-free washing medium or by denudation from their granulosa cells followed by one inhibitor-free washing step.
Stimulation of nuclear maturation was performed by placing the oocytes in IVM media supplemented with 1.5 IU/ml recombinant human chorionic gonadotropins (rhCG; Ares Serono International) and 5 ng/ml recombinant human epidermal growth factor (rEGF; Roche Diagnostics, Brussels, Belgium) for 18 h.
In all cultures, oocytes (groups of 15) were placed in microdroplets of 30 µl under oil (Sigma) and dishes were placed in a humidified atmosphere of 37°C, 5% CO2 in air.
IVF/In Vitro Culture (IVC)/Embryo Transfer (ET)
Epididymal sperm suspensions were prepared from adult mice, preincubated for 2.5 h to ensure capacitation in KSOM supplemented with 3.0% BSA fraction V (Sigma). COC or DO were inseminated into 40-µl droplets and sperm was added at a final dilution of 2 x 106/ml and incubated in a humidified atmosphere of 37°C, 5% CO2 in air for 3 h.
Inseminated oocytes were washed away from sperm by gently pipetting and were then cultured in 40-µl microdroplets of KSOM supplemented with 0.5% crystalline BSA fraction V (Eurobiochem, Louvain la Neuve, Belgium) for 3 or 4 days. The culture dishes contained groups of 15 embryos/droplet and were kept in a humidified atmosphere of 37°C, 5% O2, 5% CO2, and 90% N2.
Embryos (at Days 3 or 4 after culture) were surgically transferred into the uterine horns of 8- to 12-wk CD1, 2.5-day pseudopregnant females.
Fluorescence Staining and Analysis
COC were collected as previously described, immediately denuded, and transferred to washing medium containing 10 µM PDE3 inhibitor and 2 µg/ml Hoechst 33342 for 10 min. Small microdroplets of this medium containing 5 oocytes were placed onto a coverslip. In order to obtain a better visualization of the chromatin, the oocytes were slightly squeezed between two coverslips. Oocytes were analyzed using a fluorescence microscope equipped with filter sets for ultraviolet (Olympus, Omnilabo N.V., Aartselaar, Belgium).
Experimental Design
A schematic diagram of the study is shown in Figure 1.
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Experiment 1: Effect of PDE3 inhibitor on oocyte meiotic process and embryo viability COC were retrieved 24 h after eCG and placed in culture with or without inhibitor for 24 h. They were further processed as follows: a) To assess the effect of PDE3 inhibitor on meiosis arrest, a part of inhibitor-treated (group B) and untreated (group C) oocytes were denuded and maturation stages (GV, GVBD, and polar body [PB]) were recorded. b) To study the possible adverse effect by PDE3 inhibitor on embryo viability, oocytes were processed as follows: All COC treated or untreated were washed and stimulated for 18 h. Afterwards, the oocytes (group A, in vivo control; group B, inhibitor treated, 24-h IVM arrest + 18-h stimulation; and group C, untreated, 24-h IVM + 18-h stimulation) underwent IVF. The day after, the two-cell rates were recorded. Three days after IVC, embryos were evaluated and transferred to pseudopregnant females, and the number of newborns was recorded 18 days later.
Experiment 2: Acquisition of oocyte developmental competence by meiotic arrest COC were retrieved 24 h after eCG and placed in PDE3 inhibitor culture for 24 h. After this time, oocytes were removed from inhibitor and stimulated for 18 h. Oocytes for in vitro controls were retrieved 24 h after eCG and further stimulated for 18 h. Both were further processed as follows: a) To assess the effect of PDE3 inhibitor on the meiotic progression of COC, a part of inhibitor-treated COC and a sample of in vitro control COC were denuded after stimulation and maturation stages (GV, GVBD, and PB) were recorded (not depicted in Fig. 1). b) To assess whether IVM arrest by PDE3 inhibitor could influence the oocyte competence for subsequent embryo development, oocytes from group A' (in vivo control, 48 h), group B' (in vivo control, 24 h), group C' (inhibitor treated, 24-h IVM-arrest + 18-h stimulation;), and group D' (in vitro control 18-h stimulation) were inseminated. The day after, the two-cell rates were recorded. Four days after IVC, embryos were evaluated and transferred (groups A', C', and D') to pseudopregnant females, and the number of newborns was recorded 17 days later.
Experiment 3: Analysis of chromatin staining patterns during meiotic arrest
Spatial chromatin organization of the GV from the groups before and after nuclear arrest was analyzed. COC were retrieved 24 h after eCG and placed in culture with PDE3 inhibitor (10 µM) for 24 h. GV oocytes for controls were retrieved 24 and 48 h after eCG. Only oocytes with a diameter 
70 µm were included in this analysis.
Statistical Analysis
Differences on oocyte maturational stages were calculated by Student t-test. Differences in the proportion of two-cell, morula-, and blastocyst-stage embryos, newborns, and differences on the type of chromatin configuration in oocytes between the groups were compared by one-way ANOVA test. When ANOVA indicated a significant difference (P < 0.05), the Tukey post hoc test was performed. Percentages were statistically analyzed using arcsine-transformed data. Each experiment included at least three independent replicates. Data are presented as the mean percentages (mean %) ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of PDE3 inhibitor on oocyte meiotic process Culturing the COC with 10 µM PDE3 inhibitor (n = 139) for 24 h maintained 96.0% ± 3.2% of oocytes at GV stage compared with 15.0% ± 6.6% of the untreated COC group (n = 135) (P < 0.001). All the remaining oocytes in PDE3 inhibitor and untreated cultures had a PB extruded (4.0% ± 3.2% versus 85.0% ± 6.6%; P < 0.001).
Effect of PDE3 inhibitor on embryo viability To evaluate whether culture with 10 µM PDE3 inhibitor could affect oocyte developmental capacity and viability, IVF, IVC, and ET were performed. Results for embryo development are represented in Table 1. The use of PDE3 inhibitor for 24 h did not impair oocyte developmental capacity for two-cell formation after IVF. The proportion of two-cell embryos formed for the inhibitor-treated COC (group B) was significantly lower compared with in vivo controls (group A). The two-cell formation rate for the untreated group (group C) was very low compared with the inhibitor-treated and in vivo control groups (P < 0.01).
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The oocytes that were arrested by PDE3 inhibitor resulted in a significantly lower proportion of morula compared with in vivo controls. Considering the two-cell-stage embryos as the starting point for culture, 70.5% ± 16.8% of the inhibitor-treated oocytes (group B) developed to morula, which was not statistically different from the in vivo controls.
The untreated COC (group C) showed a deficient embryo developmental capacity compared with inhibitor-treated and in vivo controls (8.3% ± 11.3%) (P < 0.001), which was also significantly lower for the transition of two-cell embryos to morula stage.
As shown in Table 2, pharmacological arrest of oocytes by PDE3 inhibitor (10 µM) did not affect embryo viability. When compared with in vivo controls, a similar number of living embryos were obtained, excluding any toxicity by the PDE3 inhibitor on further embryo development. None of the few embryos (n = 7) obtained in the untreated culture (group C) and transferred resulted in live offspring (P < 0.001).
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Experiment 2
Capacity for meiosis progression after removal from PDE3 inhibitor COC removed 24 h after culture in Org 9935 (10 µM) and stimulated (group C'), were capable of progressing through meiosis to the same extent as the COC that underwent stimulation immediately after retrieval (group D') (Table 3). About 90.0% of oocytes in the inhibitor-treated group and about 85.0% for in vitro controls extruded the first PB 18 h after stimulation.
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Embryo developmental competence of oocytes after inhibitor removal To evaluate whether arrest by PDE3 inhibitor (10 µM) could improve oocyte developmental competence and viability compared with nonarrested oocytes, IVF, IVC, and ET were performed. Fertilization (two-cell) rates from the in vivo-ovulated controls differed according to the exposure time to eCG: It was decreased in oocytes retrieved after only 24 h compared with 48 h (P < 0.05) (Fig. 2A). Fertilization capacity of oocytes exposed in vitro to PDE3 inhibitor (group C') was 81.4% ± 3.6%. This figure equaled the in vivo-ovulated control of 24-h eCG treatment (group B'; 85.3% ± 7.9%) but is still lower compared with the 48-h eCG treatment (group A'; 98.4% ± 1.1%) (P < 0.001) (Fig. 2A). In vitro control oocytes, punctured 24 h after eCG priming (Group D'), had the lowest capacity to fertilize after IVF (57.6% ± 13.8%) (P < 0.05) (Fig. 2A).
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Embryo development of ovulated COC from 24-h eCG treatment (group B') resulted in 68.2% ± 7.9% morula stage and 66.7% ± 8.9% blastocyst stage. These values were lower than from 48-h eCG-exposed mice (group A') (91.6% ± 6.9% and 87.8% ± 8.5%; P < 0.01 and P < 0.05, respectively) (Fig. 2, B and C). From the PDE3 inhibitor-treated COC (group C'), 60.2% ± 9.7% and 57.7% ± 8.7% progressed to morula and blastocyst stages at rates similar to in vivo ovulated oocytes from 24-h eCG-exposed mice (Fig. 2, B and C). Oocytes matured in vitro without any pretreatment of COC after 24-h eCG priming (group D') had a lower capacity to develop into morula and blastocyst (42.6% ± 10.6% and 40.1% ± 12.0%) compared with the arrested oocytes (P > 0.05) and both ovulated control groups 24 h (P < 0.05) and 48 h after eCG priming (P < 0.001) (Fig. 2, B and C).
The overall success rate for obtaining live young from oocytes arrested by PDE3 inhibitor (group C') was 41.0%, where only 15.0% was obtained from the in vitro control group (Table 4). When considering only the transfers resulting in live young (implantation had surely occurred in those), live birth rate per embryo transferred from the arrested oocytes (42.3% ± 13.6%) was similar to the in vivo control group (41.1% ± 13.2%) and higher compared with in vitro control group (33.7% ± 19.2%), though not significantly.
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PDE3-arrested oocytes from immature follicles produced embryos resulting in fertile transfer cycles (7 out of 7 transfers) at every occasion, while nonarrested oocytes developed in embryos that did not lead to fertile cycles in 4 out of 7 transfers.
Experiment 3
Chromatin staining patterns in GV oocytes arrested by PDE3 inhibitor Different types of chromatin configurations were observed after Hoechst staining (n = 468): NSN, intermediate, and SN configurations. The NSN nucleolus of the GV has a more homogeneous nucleoplasm containing small and large chromatin foci (Fig. 3A). The intermediate chromatin pattern is clearly classified as a nucleus containing some diffused euchromatin and aggregates of condensed chromatin with a partial rim of perinucleolar chromatin (Fig. 3B). The SN of the GV is characterized by a nucleolus surrounded by condensed chromatin and a nucleoplasm with some threads of chromatin extending between the periphery of the nucleolus and the periphery of the nucleus (Fig. 3C).
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The meiotic arrest by PDE3 inhibitor for 24 h resulted in a similar proportion of SN configuration compared with oocytes retrieved 48 h after eCG (82.4% ± 3.2% and 90.0% ± 1.8%, respectively) (Fig. 4). These values were higher compared with those obtained 24 h after eCG (60.2% ± 14.6%; P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acquisition of Oocyte Developmental Competence After Meiotic Arrest by PDE3 Inhibitor
Oocyte development during the latter stages of follicle growth in vivo embraces GV chromatin organization, cytoplasmic microtubules organization [21], and synthesis and storage of checkpoint molecules necessary for later embryonic development [22]. Isolation of GV oocytes from antral follicles truncates the cytoplasmic maturation process by the spontaneous resumption of meiosis. An extension of the culture period by blocking meiosis progression could allow intraoocyte cytoplasmic changes and oocyte development to be completed.
Effective block of meiosis in rodent oocytes has already been demonstrated by nonspecific PDE inhibitors in vitro [1, 23] and by selective PDE3 inhibitors in vitro [16] and in vivo [19]. Although the exact regulatory mechanism of PDE3A activation in oocytes remains unclear, there is enough evidence that this protein is involved in the regulation of meiosis. In rats, PDE3A activity increases during LH-induced in vitro maturation and reaches its peak after 2 h of spontaneous in vitro maturation [24]. Selective PDE3 inhibitors, such as Org 9935, have been used to block meiosis of oocytes from large mammalians and thus has demonstrated their involvement on the regulation of oocyte maturation in these species as well [18, 25–27].
The purpose of our study was to investigate the effects of the specific PDE3 inhibitor on mammalian oocytes removed from immature antral follicles. Therefore, the oocytes were retrieved 24 h after gonadotropin injection when the antral follicles were not yet fully developed. From the present results, the addition of the specific PDE3 inhibitor, Org 9935, in the medium for 24 h maintained mouse oocytes meiotic arrested with a visible GV and without signs of oocyte deterioration. The inhibitory activity was reversible, and oocytes underwent GVBD and extruded the first polar body to the same extent as the nonarrested oocytes, showing that a reconstitution of the PDE3 activity occurred after inhibitor removal. In this study, the reverse inhibitory effect was not harmful to the oocytes as proven by the capability for fertilization and pre- and postimplantation development. Expectedly, in the first experiment, the two-cell formation rate for the untreated group was very low compared with the inhibitor-treated and in vivo control group. In the untreated oocytes, the meiotic process is reinitiated immediately upon culture, leading to postmeiotic aging of oocytes at the moment of fertilization 42 h later (24 h + 18 h stimulation). Cytoplasmic deficiencies characteristic of MII oocyte aging in culture are peripheral conglomerations or centripetal migration of cortical granules [28]. Zona hardening, which may be caused by cortical granules exocytosis [29], affects the capacity for proper fertilization [30]. Aging of oocytes at MII also causes disorganization in spindle structure and displacement of chromosomes [28, 31], leading to errors in chromosome segregation and, consequently, embryo abnormalities [32].
In the second experiment, the results showed that the two-cell rates in the PDE3 inhibitor group were higher compared with the nonarrested group and the embryo development was improved, although not significantly different. These results are consistent with the idea that, during this arrest period, the oocytes could have built up, modified, and stored newly synthesized proteins important for the further development. It could also be that the PDE3 inhibitor, by maintaining or increasing the intraoocyte cAMP concentrations, positively influences mechanisms related to oocyte competence.
Another reason for the higher two-cell rate obtained from the inhibited group could be due to a Ca2+ uptake and storage by the COC in culture. Ca2+ signals activate important cellular responses [33]. Induction of Ca2+ oscillations occurs both during exit from meiosis and during mitosis and acts by increasing the number of inner cell mass cells [34, 35]. Indeed, a high number of live young was obtained following the arrest period in our study. The relation between the PDE3 block and the Ca2+ uptake in culture by the COC during the arrest period needs to be studied.
The embryo development rates observed for the in vivo-ovulated control group (24 h after eCG + hCG) was similar compared with rates of the inhibited oocytes, therefore the 24-h inhibition could not replace all of the lacking factors caused by the short in vivo gonadotropin exposure period. It must be noted, however, that, at the end of culture (42 h) of the inhibitor-treated COC, some cumulus cells had already spontaneously lost contact with the oocyte. It is known that cumulus cells-oocyte connection during maturation affects cytoplasmic maturation and embryo developmental capacity [35]. Therefore, modifications of the culture conditions aiming for a better oocyte-cumulus contact, while maintaining the oocyte for a longer period in culture, may result in a better outcome. The implantation potential from PDE3-arrested oocytes in fertile cycles became equivalent to in vivo controls. Furthermore, all transfer cycles from the arrested oocytes were fertile, while this was not the case for in vivo-matured oocytes (2 out of 7 nonfertile transfers). Embryos from nonarrested oocytes did not lead to fertile transfers in 4 out of 7 cycles. Further study is needed to confirm whether using the meiosis-arrest technique provides embryos that can interact more favorably with endometria from pseudopregnant mice.
Several substances acting distinctively on the cAMP pathway have been used to extend the culture period [13, 36–39]. The inhibitor of protein phosphorylation, 6-dimethylaminopurine, has been shown to alter the kinetics of nuclear maturation of mice and to compromise the subsequent developmental competence [13]. More selective inhibitors of cyclin-dependent kinases (roscovitine and butyrolactone I) have been proven not to be deleterious for bovine embryo development [38], and even improvement of the development was demonstrated [39]. Nevertheless, it is questionable whether the kinase and phosphorylation inhibitors of broad or more selective spectrum might interfere with kinase activities that alter the function of proteins related to embryo development.
Chromatin Configuration in the GV Oocytes
The analysis on the chromatin configuration staging performed in this study revealed that GV oocytes retrieved from antral follicles of 48-h eCG-exposed adult mice presented some heterogeneity in their chromatin pattern: nonsurrounded (NSN), intermediate, and surrounded (SN) nucleolus. This heterogeneity was previously reported [6, 8]. The progression toward SN configuration has been associated with transition from active to inactive transcriptional activity in oocytes of mouse [8], cows [11], and humans [10]. In the present findings, it was observed that the SN population of punctured oocytes was significantly lower at 24 h compared with 48 h after eCG priming. This confirms that, concomitant to the antral development in adult primed mice, a transition from NSN to SN configuration takes place.
We observed that 48 h after eCG, about 90.0% of the oocytes had a SN constitution. Gonadotropin stimulation has been associated with an increased proportion of SN configuration in fully grown mouse oocytes [8] and with transcriptional repression [12]. Although the role of gonadotropin in regulating transcriptional activity in oocytes is not yet defined, it has been suggested that surrounding compact granulosa cells are required [12]. This is not surprising because these cells serve as a mediator of gonadotropin action. Gonadotropin activates production of cAMP [40]. In rodents, cAMP increase in the oocytes caused by FSH is dependent on patent gap junctions [41]. In somatic cells, the activation of cAMP-dependent protein kinase (PKA) has been described to result in the phosphorylation of certain transcription factors such as cAMP-response element (CRE) binding protein (CREB) and CRE modulatory protein (CREM), which bind to CR elements (cAMP responsive element) located on the promoter of many genes, leading to activation of transcription. The shorter isoforms of CREM, the inducible cAMP early repressors (ICER), which lack the kinase-transactivating domains, have been described to act as transcription repressors proteins [42]. Treatments known to increase cAMP in the rat granulosa cells induce transcription of protein repressors [43]. These findings indicate that the induction of gene repression is mediated via the PKA signal transduction pathway. It could be interesting to investigate whether the transcription repression in oocytes by gonadotropin administration is activated by similar mechanisms as in the somatic cells, via PKA signaling.
In this study, the arrest in vitro allowed the transition from NSN to SN configuration in the oocyte nucleus. The proportion of SN configuration in the GV of the 24-h-arrested oocytes was comparable with the proportion obtained 48 h after priming and significantly higher compared with 24 h after priming. It is unlikely that the PDE3 inhibitor would influence any of the mechanisms in granulosa cells due to its specificity to the oocyte [16]. Although we speculate the possibility that the PDE3 inhibitor, by blocking cAMP hydrolysis in the oocyte, could activate, via the PKA pathway, repressors of gene transcription.
During oocyte growth, gene expression is very active in order to provide enough maternal RNA to sustain maturation and early embryo development [44]. There exists evidence that, once hormonal stimulation has been carried out in vivo, the process of chromatin condensation and transcription repression in fully grown oocytes is activated [8, 12, 13] and inhibitor of protein phosphorylation (such as 6-dimethylaminopurine) does not reverse this process [13]. Therefore, a limited stimulation might be considered when collecting mammalian oocytes for IVM with a meiotic arrest period because a prolonged period of transcription inactivation is deleterious to oocytes [12].
In our study, a similar proportion of PB-extruded oocytes was encountered in the arrested group compared with the nonarrested group (experiment 2), although the latter had a higher proportion of NSN. This might be explained by the fact that, in vitro, oocytes with NSN configuration were also competent to resume meiosis by probably transforming rapidly into SN [6].
The association between SN configuration in fully grown oocytes and the acquisition of embryo developmental competence has been reported. A higher embryonic development was obtained from the SN oocytes compared with NSN oocytes [7]. In the present study, the increase of SN population in the arrested group might have contributed to the significantly higher proportion of two-cell-stage embryos compared with the nonarrested oocytes. But the embryo development for the arrested group was not as high as for the in vivo 48 h after priming, although the proportion of SN oocytes was similar. It may not be ruled out that, by culturing the arrested oocytes, there is an acceleration of the transition from the active to inactive state, with negative effect for some oocytes.
Furthermore, the in vivo-ovulated oocytes collected after the 24-h eCG exposure had a lower embryo development compared with the ones from 48-h eCG exposure. So we might consider that the class of NSN could have been ovulated or that other intraovarian and intraoocyte factors were deficient for these ovulated SN.
In conclusion, the present findings demonstrate that a temporal meiotic arrest by PDE3 inhibitor allowed synchronization within the population of immature oocytes, which was beneficial to the cytoplasmic maturation process, demonstrated by the overall improved embryonic development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: D. Nogueira, Follicle Biology Laboratory, Dutch-Speaking Free University of Brussels (Vrije Universiteit Brussel), Laarbeeklaan, 101, Brussels, 1090, Belgium. FAX: 32 2 477 50 60; lrianad{at}az.vub.ac.be
Received: 9 July 2003.
First decision: 23 July 2003.
Accepted: 7 August 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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