Hum. Reprod. Advance Access originally published online on January 23, 2008
Human Reproduction 2008 23(3):504-513; doi:10.1093/humrep/dem344
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes
1 Center for Reproductive Medicine, Department of Obstetrics and Gynecology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, People's Republic of China 2 Department of Obstetrics and Gynecology, the Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510000, People's Republic of China 3 Department of Obstetrics and Gynecology, Guangdong Provincial People's Hospital, Guangzhou 510080, People's Republic of China
4Correspondence address. IVF Program, Department of Obstetric and Gynecology, Stanford University Medical Center, Palo Alto, CA 94304, USA. E-mail: shuyimin{at}hotmail.com
These three authors contributed equally.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
BACKGROUND: In an attempt to allow for acquisition of oocyte cytoplasmic maturation, PDE3 specific inhibitor, cilostamide and adenylate cyclase activator, forskolin were used to extend pre-maturation culture of immature human oocytes.
METHODS: Cumulus–oocyte complexes retrieved from unstimulated ovaries were continuously cultured under 20 µM cilostamide or 50 µM forskolin, alone or in combination for 6, 12, 24 or 48 h, respectively. Levels of intercellular gap junction communication (GJC) and maturational status were examined at these designated time points. Metaphase II oocytes obtained following 54 h biphasic culture (with meiotic inhibitors from 0 to 24 h, no meiotic inhibitors from 24 to 54 h) were subject to intracytoplasmic sperm injection and embryos were cultured for five more days.
RESULTS: Both cilostamide and forskolin delayed spontaneous meiotic progression after continuous culture with immature human oocytes. Combined treatment of cilostamide and forskolin significantly lowered the rates of germinal vesicle breakdown (GVBD) at 6, 12, 24 or 48 h after meiotic inhibitory culture, when compared with the control (all P < 0.05). A delay of 6 h for the loss of GJC was also observed under the combined treatment of cilostamide and forskolin. The fertilization rate was significantly higher under the combined treatment of cilostamide and forskolin than that of the control. Although the rates of oocyte maturation and embryo cleavage were similar among groups, there was a slight but non-significant increase in blastocyst formation rate with the treatment of cilostamide and forskolin.
CONCLUSIONS: Combined treatment of cilostamide and forskolin positively influences oocyte developmental competence by exhibiting a synergistic effect on the prevention of GJC loss and resumption of meiosis.
Key words: human oocyte/cytoplasmic maturation/meiotic resumption/cilostamide/forskolin
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
In vitro maturation (IVM) of human oocytes is an attractive alternative to controlled ovarian stimulation in the treatment of infertility. However, embryo quality and developmental potential of in vitro matured oocytes have been shown to be compromised when compared to in vivo matured oocytes (Trounson et al., 2001
). Competent oocyte development requires synchronization between nuclear and cytoplasmic maturation. Cytoplasmic maturation, defined as the capability to support fertilization and early embryo development, comprises the accumulation of mRNA and protein, redistribution of cytoplasmic organelles and changes in cellular metabolism during pre-ovulatory follicle development. The completion of nuclear maturation does not necessarily signify the acquisition of developmental competence (Picton et al., 2002; Colleoni et al., 2004). The relevance of the lower developmental capacity of the in vitro-matured oocyte to incomplete or delayed cytoplasmic maturation has been established in different species (Damiani et al., 1996; Fulka et al., 1998; Campagna et al., 2001; Salamone et al., 2001; Schramm et al., 2003; Jimenez-Macedo et al., 2006).
In humans, it takes several days for a small antral follicle to grow to the final pre-ovulatory size (
14 mm) and achieve pre-ovulatory oocyte maturation in vivo (Branigan and Estes, 2005). However, antral follicles retrieved for IVM have undergone a substantially shorter period of growth compared to pre-ovulatory follicles retrieved for conventional IVF (Cha et al., 2000; Smith et al., 2000; Cekleniak et al., 2001), inflicting a shorter period of development on the enclosed oocytes. Owing to the lack of stimulus to overcome the inhibitory effect that substances in follicular fluid and granulosa cells cause to the oocytes, in vitro matured oocytes undergo maturation prematurely as they are still in the process of acquiring cytoplasmic maturation in vivo (Gilchrist and Thompson, 2007). Although immature oocytes can resume meiosis following isolation from antral follicles in mammals (Edwards et al., 1965), cytoplasmic maturation lags behind nuclear maturation (Janssenswillen et al., 1995; Huang et al., 1999). It is then necessary to allow more time for the immature oocyte to undergo cytoplasmic maturation and consequently acquire a higher developmental competence in vitro (Wu et al., 2006).
Although the mechanism of oocyte maturation in vivo and in vitro is still poorly understood, it has been generally accepted that the second messenger cyclic adenosine mono-phosphate (cAMP) plays a critical role in maintaining meiotic arrest in mammalian oocytes. Relatively high level of cAMP within the oocyte is essential to maintain meiotic arrest, whereas a drop in intra-oocyte concentration of cAMP enables resumption of meiosis (Sela-Abramovich et al., 2006). The intra-oocyte concentration of cAMP is regulated by the balance between the activity of two kinds of enzymes: adenylyl cyclase (AC) and phosphodiesterases (PDEs), which are responsible for cAMP synthesis and degradation, respectively (Fimia and Sassone-Corsi, 2001; Conti et al., 2002).
After being synthesized by AC in cumulus cells, the cAMP is transferred to oocyte through gap junction communication (GJC). At least 11 families of PDE isoenzymes have been identified (Manganiello et al., 1995; Soderling and Beavo, 2000); among them PDE3 and PDE4 have been shown to account for the majority (>80%) of the cAMP-hydrolyzing PDE activity in vascular smooth muscle cells (Leroy et al., 1996). Specific PDE subtypes are found to be differentially localized within the follicular somatic and germ cells compartments, with PDE4 being mainly involved in the metabolism of cAMP in granulosa cells and PDE3 in oocytes (Conti et al., 2002; Sasseville et al., 2006). The cAMP-elevating agents, including membrane permeable derivatives cAMP, specific PDE subtype inhibitors and AC activator, have been shown to inhibit or attenuate spontaneous meiotic maturation of murine (Nogueira et al., 2003a), bovine (Aktas et al., 1995; Luciano et al., 2004), porcine (Funahashi et al., 1997; Bagg et al., 2006) and human oocytes (Nogueira et al., 2003b, 2006).
There is substantial evidence showing that intimate cell-to-cell communications between follicular somatic cells and the oocyte via GJC, as well as other junctional complexes, is critical for the completion of meiotic and cytoplasmic maturation (Gilchrist and Thompson, 2007). Gap junctions provide essential nutrients and metabolic support for the oocytes; at the same time, meiotic inhibitory substances, such as cAMP, purines and other putative regulatory molecules, are also transferred from follicular cells to the oocyte to maintain meiotic arrest (Jamnongjit and Hammes, 2005). In vivo, the LH surge triggers disruption of the gap junctions between the oocyte and granulosa cells within the pre-ovulatory follicle (Eppig, 1991a). Isolation of immature oocytes from their antral follicle environment to undergo IVM might interrupt the transfer of nutrients and metabolic support essential for completion of maturation, resulting in premature activation of nuclear maturation and compromised ooplasmic maturation and subsequent development. For this reason, maintaining the physical integrity of GJC between follicular cells and the oocyte during the IVM process is essential for the acquisition of cytoplasmic maturation (Carabatsos et al., 2000).
In an attempt to improve oocyte developmental competence, controlling nuclear and cytoplasmic maturation by temporary inhibition or attenuation of meiotic progress has been applied in different mammals with more or less success. Considering the critical role of cAMP and GJC in the bidirectional communication between oocytes and granulosa cells during follicular and oocyte development, we aimed to evaluate the efficacy of type 3 PDE inhibitor (cilostamide) and adenylate cyclase activator (forskolin) on meiotic resumption and embryonic development of human immature oocytes. Dynamic changes of GJC between oocyte and cumulus cells at the condition of cilostamide and/or forskolin were also assessed at the same time.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Patient and oocyte source
This study was approved by the medical ethical committee of the First Affiliated Hospital of Sun Yat-Sen University. Informed consent was obtained from patients donating oocytes and sperm. COCs were obtained from 292 patients who underwent gynecological surgery for the diagnosis and/or treatment of gynecological disorders between June 2004 and November 2006. All oocytes were retrieved from natural cycles 3–7 days after clearance of uterine bleeding without FSH priming or hCG administration. A total of 827 COCs were collected, 362 (43.8%) were aspirated by pump from ovaries during laparoscopic gynecologic surgery and the remaining were collected from surgically removed ovaries by manual aspiration with 10 ml syringe. The mean age of patients was 34.1 ± 5.3 years. Donor sperm were used for intracytoplasmic sperm injection (ICSI).
Classification and collection of COCs
Immediately after retrieval, the COCs were classified into four types as follows: type I, compact mass of 3–5 layers of granulosa cells; type II, expanded distal layers of granulosa cells (cumulus), but a compact proximal (corona cells) granulosa cell layer; type III, expanded cumulus and corona cells; type IV, partially denuded oocytes (DOs) due to expanded granulosa cells or mechanical factors related to the aspiration procedure (Cohen et al., 1986; Smitz et al., 2001). A total of 730 COCs (88.3%) classified as of type I or type II were used in this study, with the remaining 97 type III or type IV COCs (11.7%) being discarded from this study. Type I and type II oocytes from the same patient (sibling oocytes) were randomized among groups.
Culture medium and oocyte culture
The basal culture medium was TCM-199 with 26.2 mmol NaHCO3 (Invitrogen, USA) supplemented with 100 mIU/ml FSH (Gonal-F, Serono, Geneva, Switzerland), 5 ng/ml insulin, 5 ng/ml selenium, 5 µg/ml transferrin (Invitrogen, USA), 1 µg/ml 17β-E2 (Sigma, St Louis, USA) and 10% fetal bovine serum (FBS, Gibco, USA). Medium I is the basal culture medium modified with meiotic inhibitor, otherwise Medium II is the basal culture medium modified with 150 mIU/ml LH (Serono, Geneva, Switzerland). All oocyte and embryo cultures in this study were carried out at 37°C in humidified atmosphere in an incubator gassed with 5% CO2 in air. Stock solutions for cilostamide (100 mM) and forskolin (100 mM) were prepared in dimethyl sulfoxide and protected from light at –4°C, and were diluted to the appropriate concentration immediately before their use. Cilostamide and forskolin were added at a final concentration of 20 and 50 µM, respectively. Directly after aspiration, single COC was washed in inhibitor-free flushing medium (TCM 199 with HEPES) prior to culture in 50 µl droplets consisting of Medium I under oil. COCs were randomized over four culture conditions: (i) control (without meiotic inhibitor); (ii) cilostamide alone (20 µM); (iii) forskolin alone (50 µM); (iv) cilostamide (20 µM) + forskolin (50 µM).
Experiment 1: effects of cilostamide and forskolin on cumulus–oocyte GJC and oocyte meiotic resumption
Cumulus–oocyte GJC was measured at designated time points, i.e. 6, 12, 24 or 48 h after continuous exposure to Medium I with different meiotic inhibitors. COCs cultured in Medium I without meiotic inhibitors were set as the control. The measurement was performed under fluorescence microscopy and was then calculated quantitatively as the amount of the acetoxymethyl (AM) ester derivative of the fluorescent indicator calcein in the oocytes, transferred from the cumulus cells through gap junctions via passive diffusion. The gap junction dye transfer from cumulus cells to the oocyte was measured as previously described by Thomas et al. (2004a) and briefly outlined below. After being cultured in control or meiotic inhibitors, COCs were transferred to a solution of 5 µM calcein-AM (39,69-di(O-acetyl)-29,79-bis[N,N-bis-(carboxymethyl) amino methyl]-fluorescein, tetraacetoxy methyl ester; C-3100; Molecular Probes, Eugene, OR, USA) freshly prepared in a modified phenol red- and BSA-free B-TCM with polyvinyl alcohol (0.3 mg/ml; Sigma) (CAMBTCM) with or without meiotic inhibitors. The COCs were then cultured in media with the dye with or without meiotic inhibitors for 15 min. COCs were then transferred back to their respective culture treatment conditions for 25 min to allow for dye exchange between the cumulus cells and the oocyte. Unincorporated dye was then removed by three washes in calcein-AM-free CAMBTCM with or without meiotic inhibitors. Prior to fluorescence analysis at each time point, oocytes were completely denuded from surrounding cumulus cells so that only dye confined within the DOs after transport via gap junctions was measured.
Within 30 min of denudation, intra-oocyte fluorescence emission of calcein in pulsed oocytes was measured by using a fluorophotometric-inverted microscope. Fluorescence light power and photomultiplier settings were kept constant for all experiments. A single photo with one oocyte was carried out and used for the analysis. The images and the intensity of fluorescence reading were processed by the confocal software (LSM-510 confocal laser scanning microscope, Zeiss, Oberkochen, Germany). DOs in the experimental field of view were analysed singularly and independently from neighboring oocytes. Fluorescence readings of DOs in each replicate experiment were represented as relative fluorescence intensity compared to the t = 0 h control DO reading (%).
To confirm that intra-oocyte fluorescence is dependant on and due to conducting gap junctions between the cumulus cells and the oocyte (Thomas et al., 2004a; Sela-Abramovich et al., 2006), COCs were cultured in the presence of carbenoxolone (CBX; 3b-hydroxy-11-oxoolean-12-en-30-oic acid 3-hemisuccinate; Sigma), a known gap junction blocker. Owing to the unavailability of large number of human oocytes, a total of 20 COCs were cultured with carbenoxolone at a concentration of 30 µM for 3 h. The cumulus cells were then denuded and GJC levels at the time points of 3 and 3.5 h following culture with carbenoxolone were analysed following the cumulus removal.
To investigate the capacity of meiotic inhibitors to maintain oocytes at GV stage during the pre-maturation culture with nuclear maturation inhibitors, the oocytes denuded at different times of incubation under different treatment protocols were examined under Hoffman interference inverted microscope immediately following fluorophotometry. Each oocyte was assessed for the presence of the first polar body or absence of a germinal vesicle and classified as being at either GV, metaphase I (MI), or MII stages. The oocyte is at germinal vesicle breakdown (GVBD) when a not-well defined nuclear membrane is visualized. MI oocytes were defined as those in which no GV and no first polar body were visible. Those oocytes with extrusion of the first polar body were MII. Considering that all these oocytes in Experiment 1 were exposed to calcein–AM during the cumulus–oocyte GJC assay, they were not further cultured for the evaluation of fertilization and subsequent embryonic development.
Experiment 2: effects of cilostamide on meiotic progression in the absence or presence of cumulus cells
To evaluate if the inhibitory effect of cilostamide on meiotic progression occurred independently of the presence of cumulus cells, oocytes were denuded of their cumulus cells immediately after aspiration from follicles and continuously cultured with 20 µM cilostamide for 24 h. Meiotic progression of the DOs was compared with those of COCs under the same treatment protocol. Percentages of oocytes at various stages of development were calculated.
Experiment 3: effects of cilostamide and forskolin on reversibility of meiotic arrest and oocyte developmental competence
To investigate the impact of cilostamide and forskolin on fertilization and embryonic development, a biphasic culture strategy was performed. COCs were first cultured in Medium I with either cilostamide alone, forskolin alone, or cilostamide + forskolin from 0 to 24 h, after which Medium I with meiotic inhibitors was replaced by Medium II and COCs were cultured in Medium II from 24 to 54 h without meiotic inhibition. For the control, COCs were continuously cultured in Medium II for 30 h without exposure to meiotic inhibitors. The COCs were then incubated with 80 IU/ml hyaluronidase for less than 1 min as per our routine protocol for ICSI, followed by denudation of surrounding cumulus cells by stepwise mechanical stripping. Maturational status of DOs was evaluated and MII oocytes were subject to ICSI. The injected oocytes were then rinsed and transferred to 15 µl drops of pre-equilibrated Quinn’s Cleavage Medium with 5% (V/V) human serum albumin (HSA) (CooperSurgical Inc, CT, USA) under mineral oil. A fertilization check was performed from 16 to 18 h post ICSI. Fertilized oocytes with two clear pronuclei (2PN) were cultured for another 24 h to evaluate cleavage. On Day 3, the cleaved embryos were transferred to Quinn’s Blastocyst Medium (CooperSurgical Inc, CT, USA) with 5% (V/V) HSA and cultured for three more days for blastocyst formation.
Statistical analysis
Differences in the proportion of oocytes that had progressed to the various meiotic stages (GV, MI, MII) were examined using Pearson chi-square analysis with or without Yates’continuity correction as appropriate. Differences in the levels of GJC between the oocytes and the surrounding CCs, as indicated by measurement of intra-oocyte fluorescence intensity, over time and in response to treatment, were assessed using two-way analysis of variance (ANOVA). Differences in the proportion of cleaved embryos that had progressed to the blastocyst stage were examined using Pearson chi-square analysis with or without Yates’continuity correction as appropriate. Probabilities of <0.05 were considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Effect of cilostamide and forskolin on cumulus–oocyte GJC
Our experiment validated the permeation of calcein through GJC between cumulus cells and human oocyte into ooplasm by culturing the COCs with 30 µM carbenoxolone. No significant difference in GJC level was observed between the time points 3 and 3.5 h following culture with carbenoxolone (0.181 ± 0.062 versus 0.176 ± 0.054), indicating no loss or accumulation of calcein in the oocyte after denudation. In addition, the GJC level at time point 3.5 h after carbenoxolone incubation was also found to be similar to that of 24 h in the control (0.150 ± 0.046), when GJC was almost completely lost.
The oocyte–cumulus GJC assay was performed in 334 COCs cultured at control conditions or under the treatment of cilostamide and/or forskolin at different time points. In the control group, the level of GJC between the oocyte and the cumulus cells fell sharply from 100% at 0 h to 35.6% at 6 h of culture, after which the GJC level decreased stably until 24 h of culture (15.0%). The levels of GJC were similar between control and treatment groups at the time points of 24 and 48 h (Table I), suggesting that the majority of GJC loss happened in the first 24 h of culture in all groups.
|
GJC levels at the time points of 6 and 12 h of continuous exposure to meiotic inhibitors were significantly higher in the three treatment groups than in the controls except between the control and cilostamide at 12 h. As shown in Table I, treatment with cilostamide alone or forskoline alone attenuated the loss of GJC from 0 h until 24 h of culture. Compared with cilostamide, treatment of COCs with forskolin was more effective in delaying the loss of GJC at 6 h (P < 0.01). Cilostamide enhanced the effect of forskolin on cumulus cell–oocyte GJC until 12 h of culture (P < 0.01). When analysing the time-dependent change of levels of GJC, we found that the GJC level in the cilostamide + forskolin group was 2-fold greater that that of control at time points of 6 and 12 h (P < 0.01). Moreover, the GJC level of cilostamide + forskolin group at 12 h of culture was even higher than the level of control group at 6 h of culture, indicating that the combined treatment of cilostamide and forskolin had delayed the loss of cumulus–oocyte GJC for more than 6 h.
Inhibition of spontaneous nuclear maturation by cilostamide and forskolin
Combined treatment of cilostamide and forskolin significantly lowered the rates of GVBD at designated time points, when compared with controls. As shown in Fig. 1 at 6 h after culture, 45% (10/22) of cultured oocytes had GVBD in the control group, which was significantly higher than those cultured with cilostamide alone, forskolin alone or cilostamide + forskolin (10%, 2/20; 10%, 2/20; and 0, 0/20, respectively). A similar trend was observed at 12 h after culture, at this point, 18% (4/22), 40% (8/20) and 9% (2/22) of oocytes underwent GVBD when cultured with cilostamide alone, forskolin alone and cilostamide + forskolin, respectively, in contrast to 60% (12/20) in the control group. By 24 h of culture, the percentages of GVBD had increased to 40% (8/20), 50% (10/20), 41% (9/22) and 73% (16/22) for cilostamide alone, forskolin alone, cilostamide + forskolin and the control, respectively. At 48 h after culture, the GVBD rates were 60% (12/20), 80% (16/20), 45% (10/22) and 86% (19/22) for cilostamide alone, forskolin alone, cilostamide + forskolin and the control, respectively. Significantly lower rates of GVBD were obtained at 6, 12, 24 or 48 h of culture under the combined treatment of cilostamide and forskolin when compared to the control (all P < 0.05) (Fig. 1).
|
When compared with 50 µM forskolin, 20 µM cilostamide seemed more effective in maintaining oocytes at GV stage although significant difference could not be demonstrated (Fig. 1). However, forskolin amplified the inhibitory effect of cilostamide on the progression of maturation, such that none of the oocytes had undergone GVBD after 6 h culture. Treatment with 20 µM cilostamide + 50 µM forskolin delayed the onset of GVBD until 12 h after culture, at this point, 9% (2/22) COCs had undergone GVBD compared to 60% (12/20) of the control (P < 0.01).
Oocytes that had undergone GVBD progressed to MI or MII stages over time. By 12 h after continuous meiotic inhibition, none of the oocytes had extruded the first polar body in the three treatment groups and only 1 out of 20 (5%) oocytes had matured in control (Fig. 2A). Since some of the MI oocytes continued to progress through MII stage, the percentages of MI oocytes started to decline at 24 h after culture and onward in control and forskolin alone (Fig. 2B). By 48 h after culture, the percentages of MII oocytes with extrusion of the first polar body was 20% (4/20), 40% (8/20), 18% (4/22) and 64% (14/22) for cilostamide alone, forskolin alone, cilostamide + forskolin and control, respectively (Fig. 2A), with a significantly higher proportion of oocytes matured and a lower proportion of oocytes maintained at the GV stage being obtained in the control when compared to cilostamide alone or cilostamide + forskolin group (both P < 0.01) (Fig. 2C).
|
Effects of cilostamide on meiotic progression in the absence or presence of cumulus cells
As shown in Fig. 3, no significant difference was observed in the percentages of oocytes that underwent GVBD (43.6 and 37.5%, respectively) and extruded the first polar body (MII stage; 12.8 and 9.4%, respectively) in denuded and COCs groups under the continuous exposure to 20 µM cilostamide.
|
Effects of cilostamide and forskolin on reversibility of meiotic arrest and oocyte developmental competence
For a total of 186 oocytes matured out of 305 COCs in control or under a biphasic culture (with meiotic inhibitors from 0 to 24 h, no meiotic inhibitors from 24 to 54 h) in three treatment groups, the maturation rate was 61.0%. As shown in Table II, the percentages of oocytes at GV, MI, MII stages and those degenerated oocytes were not statistically different between the three treatment groups and control (P = 0.9385), indicating that the inhibitory effect of cilostamide and/or forskolin on spontaneous meiotic progression was completely reversible.
|
Table III summarizes the fertilization and embryonic development after 6 days culture subsequent to IVM. The fertilization rate was found to be significantly higher (P < 0.05) when oocytes were cultured under the treatment of forskolin + cilostamide than in the control group. As for embryonic development, similar rates of fertilized oocytes cleaved among groups. There was a trend to higher rates of embryos with optimal cleavage (4-cell on Day 2 and subsequently 8-cell on Day 3) in three treatment groups compared to control. Higher blastocyst formation rates per MII oocyte or per cleaved 2PN were obtained in the cilostamide + forskolin group as compared with the control, although significant differences were not reached (P = 0.1965, 0.066, respectively).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Direct evidence has shown that decreased oocyte developmental competence is associated with deficiencies in the mRNA storage during the oocyte growth in the ovine (Leoni et al., 2007
). In primates, oocyte growth is completed in early antral follicles, but oocyte cytoplasmic maturation is completed in later antral follicles (Schramm et al., 1999). The oocytes in this study are highly likely to be fully grown, but still acquiring cytoplasmic competence as they were collected from mid-sized antral follicles (4–10 mm). To allow for the completion of cytoplasmic maturation, COCs were cultured with specific PDE3 inhibitor, cilostamide and an AC activator, forskolin. Our results showed that meiotic progression was delayed by cilostamide and forskolin, alone or in combination, without negatively affecting embryonic development (Fig. 4).
|
The availability and use of subtype-specific PDE inhibitors provide a new opportunity for more extensive examination of oocyte maturation mechanisms and represent new and powerful experimental tools for investigating oocyte–follicular cell interactions during oocyte maturation (Thomas et al., 2002
). As a specific inhibitor of PDE3, cilostamide inhibits the degradation of intra-oocyte cAMP. While the concentration of intra-oocyte cAMP was not addressed in the present study, PDE 3 inhibitors can increase the concentration of intracellular cAMP as shown in previous reports by others (Tsafriri et al., 1996; Thomas et al., 2002; Sasseville et al., 2006). In this study, about 60% of oocytes remained blocked in the GV stage by 24 h of culture with 20 µM cilostamide, which is lower than that in a recent report by Nogueira et al. (2006), wherein 99% of cultured oocytes had an intact GV at 24 h with PDE 3A inhibitor, Org 9935. Similarly, a recent report by Vanhoutte et al. (2007) showed that >80% of the cultured oocytes were maintained at the GV stage with the treatment of cilostamide (1 and 10 µM).
The between-study discrepancies may be explained by differences in the source of immature oocytes, culture medium constituents and meiotic inhibitors. In the current study, immature oocytes were retrieved from unstimulated antral follicles with heterogeneous size, whereas those of Nogueira et al. (2006) and Vanhoutte et al. (2007) were from stimulated cycles. Their reactions to meiotic inhibitors might also differ. A critical difference between the current and above studies is the dose of FSH in basal media. As an additive to oocyte maturation medium, FSH not only induces in vitro nuclear maturation (Eppig, 1991b) and cumulus expansion (Singh et al., 1993; Calder et al., 2003), but also improves oocyte cytoplasmic maturation (Izadyar et al., 1998) and embryonic development (Alberio and Palma, 1998; Atef et al., 2005). When COCs were maintained in meiotic arrest with a suboptimal inhibitory concentration of cAMP, FSH first augmented the inhibition of GVBD by stimulating cAMP production in cumulus cells, and later became stimulatory to maturation (Downs and Eppig, 1985; Downs et al., 1988). The meiosis-arresting effect of 3-isobutyl-1-methylxanthine (IBMX), Hypoxanthine, and PDE3 inhibitor (milrinone), has been shown to be overcome by the addition of FSH in the IVM media (Downs et al., 1988; Ryan et al., 2002; Thomas et al., 2004b). Thus, higher concentration of FSH (100 mIU/ml) in current study could account for the decreased levels of GVBD inhibition because cilostamide and Org 9935 have an identical IC50 of 0.1 µM on mouse oocytes (Wiersma et al., 1998). As described in the methods, an oocyte was defined at GVBD when a not-well defined nuclear membrane was visualized. It is possible that some oocytes with unclear GV were counted as MI due to the limitation of the conventional light microscope. In addition, the lag time between oocyte collection and replacement in culture with meiotic inhibitors should also be considered.
Cumulus cells play a critical role in maintaining oocyte meiotic arrest by synthesizing cAMP and transferring it through GJC to the oocyte. This is the first report that meiotic progression of human oocytes was delayed by forskolin at a concentration of 50 µM, which is consistent with data from rodents (Dekel et al., 1984; Racowsky, 1984), porcine (Racowsky, 1985; Xia et al., 2000), and bovine (Thomas et al., 2004a, b). As discussed in the introduction, cAMP levels are differentially regulated within the oocyte and somatic cell compartments of the ovarian follicle. In this study, cilostamide exerted meiotic inhibitory effects regardless of the presence or absence of cumulus cells. While a recent report has established the functional presence of PDE3A in porcine cumulus cells (Sasseville et al., 2007), our result supports the findings in rodents (Richard et al., 2001; Shitsukawa et al., 2001), bovine (Thomas et al., 2002) and human (Nogueira et al., 2003b), that the oocyte itself, but not cumulus cells, contains an active PDE3. On the other hand, significant increase of intra-oocyte cAMP was observed after forskolin treatment of cumulus-enlcosed oocytes but not of cumulus-free oocytes (Dekel et al., 1984), indicating that cumulus cells are the main source of cAMP of oocytes. Although different mechanisms are applied, our results showed that the inhibitory effect was augmented when forskolin was supplemented with cilostamide. Ours and other published reports, therefore, suggest that forskolin had an additive effect on the inhibitory action on oocyte maturation, which is also the basis of the combined treatment of PDE3 inhibitor and AC activator.
Disruption of GJC within the ovarian follicle promotes the resumption of meiosis, either in vivo or in vitro (Sela-Abramovich et al., 2006). In bovine, GJC had reduced to 29% of that initially measured and 75% of oocytes had undergone GVBD by 7 h of in vitro culture (Thomas et al., 2004a). In porcine, a significant positive correlation was also observed between the proportion of oocytes undergoing GVBD and the proportion of cumulus–oocyte complexes that had lost gap junctions (Isobe et al., 1998; Isobe and Terada, 2001). In this study, the levels of GJC were determined by the changes of the intra-oocyte concentration of fluorescent dye, calcein. Our results demonstrated that there was a dramatic loss of GJC to 35.6 and 19.9% of that initially measured by 6 and 12 h of IVM of human oocytes in control, respectively. This loss was attenuated in response to 20 µM cilostamide and 50 µM forskolin, alone or in combination, implying the positive effect of cAMP-elevating agents on the maintenance of GJC during pre-maturation culture (Thomas et al., 2004a, b). Although the exact amount of time period required for adequate cytoplasmic maturation remains to be determined, beneficial effects of maintenance of GJC during IVM process on the promotion of cytoplasmic maturation and subsequent embryonic development have been observed in different species (Hashimoto et al., 1998; Geshi et al., 2000; Tanghe et al., 2002).
The oocyte was previously considered to be a passive recipient of the nutritional support from companion granulosa cells; however, recent studies have indicated an active role for the oocyte in controlling metabolic activity in granulosa cells (Sugiura and Eppig, 2005). Intracellular cAMP has been shown to be a regulator of GJC in various cell types and tissues (Cruciani and Mikalsen, 2002). In bovine, the intra-oocyte cAMP plays a physiological role on GJC mediated bidirectional communication between oocyte and cumulus cells (Luciano et al., 2004; Thomas et al., 2004b). For this reason, it is assumed that the prolongation of GJC between oocyte and cumulus cells after the treatment of cilostamide and forskolin could be attributable to the accumulation of cAMP in cumulus cells and oocytes. The delayed loss of GJC in turn increased the concentration of intra-oocyte cAMP, which finally delayed the progression of GVBD (Luciano et al., 2004; Thomas et al. 2004a, b). Moreover, the combined treatment of cilostamide and forskolin was more effective in delaying the loss of GJC than either treatment of cilostamide alone or forskolin alone, indicating the extent of the loss of GJC might be associated with the level of intra-oocyte cAMP.
Artificial regulation of meiotic resumption by cAMP-elevating agents has been shown to improve oocyte competence (Funahashi et al., 1997; Luciano et al., 1999; Nogueira et al., 2003a, b; Thomas et al., 2004b; Bagg et al., 2006) or have no detrimental effect (Luciano et al., 2004; Nogueira et al., 2006) on subsequent embryonic development. In this study, a significantly higher fertilization rate was achieved when COCs were treated with the combination of cilostamide and forskolin for 24 h as compared with control. Although no significant difference was demonstrated by our data, there is a trend to improved embryonic development for oocytes treated with combined cilostamide and forskolin. In support of our findings, temporary nuclear arrest of human GV oocytes with PDE3-I has proved to be beneficial for obtaining normal spindle and chromosome configurations after IVM (Vanhoutte et al., 2007).
Extending the length of the pre-maturation period allows additional time needed for acquisition of developmental competence theoretically; however, no further beneficial effect on embryonic development has been observed under a pre-maturation treatment of 48 h (Coy et al., 2005; Nogueira et al., 2006). Coy et al. (2005) compared the embryonic development after culture of oocytes in S-roscovitine; the proportions of oocytes that cleaved and developed to the blastocyst stage were higher at 24 h than in those cultured for 0 or 48 h. Similarly, a 24 h pre-maturation period proved to be more effective in preserving embryonic integrity when compared to a 48 h pre-maturation period in human (Nogueira et al., 2006). Since the refreshment of culture medium during pre-maturation period was not disclosed in the above studies, it is still unknown whether no additive improvement of embryonic development after extended pre-maturation culture (48 h) is due to the depleted nutrients or diminished activity of meiotic inhibitor over time or oocyte maturation in vitro per se.
As demonstrated by Son et al. (2005), more than 60% of human oocytes achieved nuclear maturation within 24–30 h after maturation culture and only 14% more between 48–52 h. Although there has not been an agreement regarding the optimal time interval between oocyte maturation in vitro for MII and insemination, immature oocytes cultured as late as until 48 h of IVM are prone to oocyte senescence (Ducibella, 1998). Compared to Nogueira et al. (2006), where no embryos showed optimal cleavage after up to 48 h maturation culture following 24–48 h meiotic inhibition, our results showed that 3.8–8.8% of cleaved embryos showed optimal cleavage following biphasic culture. This could be explained, in part, by a shorter maturation culture period (30 h) post meiotic inhibition because delaying sperm injection until 48 h of culture could lead to the loss of a temporal window for optimal fertilization and a compromise in subsequent embryonic development (Chian and Tan, 2002).
Taken together, our results showed that the meiotic progression of immature human oocytes could be reversibly attenuated by 20 µM cilostamide or 50 µM forskolin during the pre-maturation period. Furthermore, there is a synergistic effect between cilostamide and forskolin on the prevention of GJC loss and resumption of meiosis. Although slightly but not significantly enhanced embryonic development was obtained from our preliminary data, our results advanced the understanding of the oocyte maturation process and the factors regulating it. To optimize the IVM conditions, further studies should be performed to investigate the optimal agents and dose of meiotic inhibitor during the IVM process.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
This study was supported by the Grants from National Natural Science Foundation of China (NSFC 30271367; 30300372; 30571956) and Science Foundation of Guangdong Province (021874, 2006B35901001).
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
We are grateful for the help of medical staff, Department of Obstetric & Gynecology, The First Affiliated Hospital, Sun Yat-Sen University, for collecting the immature oocytes. We also thank Prof. William SB Yeung, Department of Obstetric & Gynecology, Queen Mary Hospital, Hong Kong University, for critical advice to this paper.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Aktas H, Wheeler MB, First NL, Leibfried-Rutledge ML. Maintenance of meiotic arrest by increasing [cAMP]i may have physiological relevance in bovine oocytes. J Reprod Fertil (1995) 105:237–245.
Alberio R, Palma GA. Development of bovine oocytes matured in a defined medium supplemented with a low concentration of r-hFSH. Theriogenology (1998) 49:195.[CrossRef][Web of Science]
Atef A, François P, Christian V, Marc-André S. The potential role of gap junction communication between cumulus cells and bovine oocytes during in vitro maturation. Mol Reprod Dev (2005) 71:358–367.[CrossRef][Web of Science][Medline]
Bagg MA, Nottle MB, Grupen CG, Armstrong DT. Effect of dibutyryl cAMP on the cAMP content, meiotic progression, and developmental potential of in vitro matured pre-pubertal and adult pig oocytes. Mol Reprod Dev (2006) 73:1326–1332.[CrossRef][Web of Science][Medline]
Branigan EF, Estes A. Use of micro-dose human chorionic gonadotropin (hCG) after clomiphene citrate (CC) to complete folliculogenesis in previous CC-resistant anovulation. Am J Obstet Gynecol (2005) 192:1890–1894.[CrossRef][Web of Science][Medline]
Calder MD, Caveney AN, Smith LC, Watson AJ. Responsiveness of bovine cumulus-oocyte complexes (COC) to porcine and recombinant human FSH, and the effect of COC quality on gonadotropin receptor and Cx43 marker gene mRNAs during maturation in vitro. Reprod Biol Endocrinol (2003) 11:14–25.
Campagna C, Sirard MA, Ayotte P, Bailey JL. Impaired maturation, fertilization, and embryonic development of porcine oocytes following exposure to an environmentally relevant organochlorine mixture. Biol Reprod (2001) 65:554–560.
Carabatsos MJ, Sellitto C, Goodenough DA, Albertini DF. Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence. Dev Biol (2000) 226:167–179.[CrossRef][Web of Science][Medline]
Cekleniak NA, Combelles CM, Ganz DA, Fung J, Albertini DF, Racowsky C. A novel system for in vitro maturation of human oocytes. Fertil Steril (2001) 75:1185–1193.[CrossRef][Web of Science][Medline]
Cha KY, Han SY, Chung HM, Choi DH, Lim JM, Lee WS, Ko JJ, Yoon TK. Pregnancies and deliveries after in vitro maturation culture followed by in vitro fertilization and embryo transfer without stimulation in women with polycystic ovary syndrome. Fertil Steril (2000) 73:978–983.[CrossRef][Web of Science][Medline]
Chian RC, Tan SL. Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles. Reprod Biomed Online (2002) 5:125–132.[Medline]
Cohen J, Avery S, Campbell S, Mason B, Riddle A, Sharma V. Follicular aspiration using a syringe suction system may damage the zona pellucida. J Assist Reprod Genet (1986) 3:224–226.[CrossRef]
Colleoni S, Luciano AM, Gandolfi F. Cumulus-oocyte communications in the horse: role of the breeding season and of the maturation medium. Reprod Domest Anim (2004) 39:70–75.[CrossRef][Web of Science][Medline]
Conti M, Andersen CB, Richard F, Mehats C, Chun SY, Horner K, Jin C, Tsafriri A. Role of cyclic nucleotide signaling in oocyte maturation. Mol Cell Endocrinol (2002) 187:153–159.[CrossRef][Web of Science][Medline]
Coy P, Romar R, Ruiz S, Canovas S, Gadea J, Garcia Vazquez F, Matas C. Birth of piglets after transferring of in vitro-produced embryos pre-matured with R-roscovitine. Reproduction (2005) 129:747–755.
Cruciani V, Mikalsen SO. Connexins, gap junctional intercellular communication and kinases. Biol Cell (2002) 94:433–443.[CrossRef][Web of Science][Medline]
Damiani P, Fissore RA, Cibelli JB, Long CR, Balise JJ, Robl JM, Duby RT. Evaluation of developmental competence, nuclear and ooplasmic maturation of calf oocytes. Mol Reprod Dev (1996) 45:521–534.[CrossRef][Web of Science][Medline]
Dekel N, Aberdam E, Sherizly I. Spontaneous maturation in vitro of cumulus-enclosed rat oocytes is inhibited by forskolin. Biol Reprod (1984) 31:244–250.[Abstract]
Downs SM, Eppig JJ. A follicular fluid component prevents gonadotropin reversal of cyclic adenosine monophosphate dependent meiotic arrest in murine oocytes. Gamete Res (1985) 11:83–97.[CrossRef][Web of Science]
Downs SM, Daniel SA, Eppig JJ. Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal gowth factor: evidence for a positive stimulus of somatic cell origin. J Exp Zool (1988) 245:86–96.[CrossRef][Web of Science][Medline]
Ducibella T. Biochemical and cellular insights into the temporal window of normal fertilization. Theriogenology (1998) 49:53–65.[CrossRef][Web of Science][Medline]
Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey, and human ovarian oocytes. Nature (1965) 208:349–351.[CrossRef][Medline]
Eppig JJ. Intercommunication between mammalian oocytes and companion somatic cells. BioEssays (1991) a 13:569–574.[CrossRef][Web of Science][Medline]
Eppig JJ. Maintenance of meiotic arrest and the induction of oocyte maturation in mouse oocyte-granulosa cell complexes developed in vitro from preantral follicles. Biol Reprod (1991) b 45:824–830.[Abstract]
Fimia GM, Sassone-Corsi P. Cyclic AMP signalling. J Cell Sci (2001) 114:1971–1972.
Fulka JJ, First NL, Moor RM. Nuclear and cytoplasmic determinants involved in the regulation of mammalian oocyte maturation. Mol Hum Reprod (1998) 4:41–49.
Funahashi H, Cantley TC, Day BN. Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization. Biol Reprod (1997) 57:49–53.[Abstract]
Geshi M, Takenouchi N, Yamauchi N, Nagai T. Effects of sodium pyruvate in nonserum maturation medium on maturation, fertilization, and subsequent development of bovine oocytes with or without cumulus cells. Biol Reprod (2000) 63:1730–1734.
Gilchrist RB, Thompson JG. Oocyte maturation: emerging concepts and technologies to improve developmental potential in vitro. Theriogenology (2007) 67:6–15.[CrossRef][Web of Science][Medline]
Hashimoto S, Saeki K, Nagao Y, Minami N, Yamada M, Utsumi K. Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes. Theriogenology (1998) 49:1451–1463.[CrossRef][Web of Science][Medline]
Huang FJ, Chang SY, Tsai MY, Lin YC, Kung FT, Wu JF, Lu YJ. Relationship of the human cumulus-free oocyte maturational profile with in vitro outcome parameters after intracytoplasmic sperm injection. J Assist Reprod Genet (1999) 16:483–487.[CrossRef][Web of Science][Medline]
Isobe N, Maeda T, Terada T. Involvement of meiotic resumption in the disruption of gap junctions between cumulus cells attached to pig oocytes. J Reprod Fertil (1998) 113:167–172.
Isobe N, Terada T. Effect of the factor inhibiting germinal vesicle breakdown on the disruption of gap junctions and cumulus expansion of pig cumulus-oocyte complexes cultured in vitro. Reproduction (2001) 121:249–257.[Abstract]
Izadyar F, Zeinstra E, Bevers MM. Follicle-stimulating hormone and growth hormone act differently on nuclear maturation while both enhance developmental competence of in vitro matured bovine oocytes. Mol Reprod Dev (1998) 51:339–345.[CrossRef][Web of Science][Medline]
Jamnongjit M, Hammes SR. Oocyte maturation: the coming of age of a germ cell. Semin Reprod Med (2005) 23:234–241.[CrossRef][Web of Science][Medline]
Janssenswillen C, Nagy ZP, Van Steirteghem A. Maturation of human cumulus-free germinal vesicle-stage oocytes to metaphase II by coculture with monolayer Vero cells. Hum Reprod (1995) 10:375–378.
Jimenez-Macedo AR, Izquierdo D, Urdaneta A, Anguita B, Paramio MT. Effect of roscovitine on nuclear maturation, MPF and MAP kinase activity and embryo development of prepubertal goat oocytes. Theriogenology (2006) 65:1769–1782.[CrossRef][Web of Science][Medline]
Leoni GG, Bebbere D, Succu S, Berlinguer F, Mossa F, Galioto M, Bogliolo L, Ledda S, Naitana S. Relations between relative mRNA abundance and developmental competence of ovine oocytes. Mol Reprod Dev (2007) 74:249–257.[CrossRef][Web of Science][Medline]
Leroy MJ, Degerman E, Taira M, Murata T, Wang LH, Movsesian MA, Meacci E, Manganiello VC. Characterization of two recombinant PDE3 (cGMP-inhibited cyclic nucleotide phosphodiesterase) isoforms, RcGIP1 and HcGIP2, expressed in NIH 3006 murine fibroblasts and Sf9 insect cells. Biochemistry (1996) 35:10194–10202.[CrossRef][Web of Science][Medline]
Luciano AM, Pocar P, Milanesi E, Modina S, Rieger D, Lauria A, Gandolfi F. Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization. Mol Reprod Dev (1999) 54:86–91.[CrossRef][Web of Science][Medline]
Luciano AM, Modina S, Vassena R, Milanesi E, Lauria A, Gandolfi F. Role of intracellular cyclic adenosine 3',5'-monophosphate concentration and oocyte-cumulus cells communications on the acquisition of the developmental competence during in vitro maturation of bovine oocyte. Biol Reprod (2004) 70:465–472.
Manganiello VC, Murata T, Taira M, Belfrage P, Degerman E. Diversity in cyclic nucleotide phosphodiesterase isoenzyme families. Arch Biochem Biophys (1995) 322:1–13.[CrossRef][Web of Science][Medline]
Nogueira D, Cortvrindt R, De Matos DG, Vanhoutte L, Smitz J. Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro. Biol Reprod (2003) a 69:2045–2052.
Nogueira D, Albano C, Adriaenssens T, Cortvrindt R, Bourgain C, Devroey P, Smitz J. Human oocytes reversibly arrested in prophase I by phosphodiesterase type 3 inhibitor in vitro. Biol Reprod (2003) b 69:1042–1052.
Nogueira D, Ron-El R, Friedler S, Schachter M, Raziel A, Cortvrindt R, Smitz J. Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development. Biol Reprod (2006) 74:177–184.
Picton HM. Oocyte maturation in vitro. Curr Opin Obstet Gynecol (2002) 14:295–302.[CrossRef][Web of Science][Medline]
Racowsky C. Effect of forskolin on the spontaneous maturation and cyclic AMP content of rat oocyte-cumulus complexes. J Reprod Fertil (1984) 72:107–116.
Racowsky C. Effect of forskolin on maintenance of meiotic arrest and stimulation of cumulus expansion, progesterone and cyclic AMP production by pig oocyte-cumulus complexes. J Reprod Fertil (1985) 74:9–21.
Richard FJ, Tsafriri A, Conti M. Role of phosphodiesterase type 3A in rat oocyte maturation. Biol Reprod (2001) 65:1444–1451.
Ryan NK, Woodhouse CM, Van der Hoek KH, Gilchrist RB, Armstrong DT, Norman RJ. Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation. Biol Reprod (2002) 66:1548–1554.
Salamone DF, Damiani P, Fissore RA, Robl JM, Duby RT. Biochemical and developmental evidence that ooplasmic maturation of prepubertal bovine oocytes is compromised. Biol Reprod (2001) 64:1761–1768.
Sasseville M, Cote N, Guillemette C, Richard FJ. New insight into the role of phosphodiesterase 3A in porcine oocyte maturation. BMC Dev Biol (2006) 6:47–54.[CrossRef][Medline]
Sasseville M, Cote N, Vigneault C, Guillemette C, Richard FJ. 3'5'-cyclic adenosine monophosphate-dependent up-regulation of phosphodiesterase type 3A in porcine cumulus cells. Endocrinology (2007) 148:1858–1867.
Schramm RD, Bavister BD. A macaque model for studying mechanisms controlling oocyte development and maturation in human and non-human primates. Hum Reprod (1999) 14:2544–2555.
Schramm RD, Paprocki AM, VandeVoort CA. Causes of developmental failure of in-vitro matured rhesus monkey oocytes: impairments in embryonic genome activation. Hum Reprod (2003) 18:826–833.
Sela-Abramovich S, Edry I, Galiani D, Nevo N, Dekel N. Disruption of gap junctional communication within the ovarian follicle induces oocyte maturation. Endocrinology (2006) 147:2280–2286.
Shitsukawa K, Andersen CB, Richard FJ, Horner AK, Wiersma A, van Duin M, Conti M. Cloning and characterization of the cyclic guanosine monophosphate-inhibited phosphodiesterase PDE3A expressed in mouse oocyte. Biol Reprod (2001) 65:188–196.
Singh B, Barbe GJ, Armstrong DT. Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte-cumulus cell complexes in vitro. Mol Reprod Dev (1993) 36:113–119.[CrossRef][Web of Science][Medline]
Smith SD, Mikkelsen A, Lindenberg S. Development of human oocytes matured in vitro for 28 or 36 hours. Fertil Steril (2000) 73:541–544.[CrossRef][Web of Science][Medline]
Smitz J, Nogueira D, Cortvrindt R, de Matos D. Oocyte in vitro maturation. In: Assisted Reproductive Techniques Laboratory and Clinical Perspectives—Gardner DK, Weissman A, Howles C, Shoham Z, eds. (2001) vol. 1. London: Martin Dunitz-The Livery House. 107–137.
Soderling SH, Beavo JA. Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions. Curr Opin Cell Biol (2000) 12:174–179.[CrossRef][Web of Science][Medline]
Son WY, Lee SY, Lim JH. Fertilization, cleavage and blastocyst development according to the maturation timing of oocytes in in vitro maturation cycles. Hum Reprod (2005) 20:3204–3207.
Sugiura K, Eppig JJ, Society for Reproductive Biology Founders’ Lecture 2005. Control of metabolic cooperativity between oocytes and their companion granulosa cells by mouse oocytes. Reprod Fertil Dev (2005) 17:667–674.[CrossRef][Medline]
Tanghe S, Van Soom A, Nauwynck H, Coryn M, de Kruif A. Minireview: functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Mol Reprod Dev (2002) 61:414–424.[CrossRef][Web of Science][Medline]
Thomas RE, Armstrong DT, Gilchrist RB. Differential effects of specific phosphodiesterase isoenzyme inhibitors on bovine oocyte meiotic maturation. Dev Biol (2002) 244:215–225.[CrossRef][Web of Science][Medline]
Thomas RE, Armstrong DT, Gilchrist RB. Bovine cumulus cell-oocyte gap junctional communication during in vitro maturation in response to manipulation of cell-specific cyclic adenosine 3',5'-monophosophate levels. Biol Reprod (2004) a 70:548–556.
Thomas RE, Thompson JG, Armstrong DT, Gilchrist RB. Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity. Biol Reprod (2004) b 71:1142–1149.
Trounson A, Anderiesz C, Jones G. Maturation of human oocytes in vitro and their developmental competence. Reproduction (2001) 121:51–75.[Abstract]
Tsafriri A, Chun SY, Zhang R, Hsueh AJW, Conti M. Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells-studies using selective phosphodiesterase inhibitors. Dev Biol (1996) 178:393–402.[CrossRef][Web of Science][Medline]
Vanhoutte L, De Sutter P, Nogueira D, Gerris J, Dhont M, Van der Elst J. Nuclear and cytoplasmic maturation of in vitro matured human oocytes after temporary nuclear arrest by phosphodiesterase 3-inhibitor. Hum Reprod (2007) 22:1239–1246.
Wiersma A, Hirsch B, Tsafriri A, Hanssen RG, Van de Kant M, Kloosterboer HJ, Conti M, Hsueh AJ. Phosphodiesterase 3 inhibitors suppress oocyte maturation and consequent pregnancy without affecting ovulation and cyclicity in rodents. J Clin Invest (1998) 102:532–537.[Web of Science][Medline]
Wu D, Cheung QC, Wen L, Li J. A growth-maturation system that enhances the meiotic and developmental competence of porcine oocytes isolated from small follicles. Biol Reprod (2006) 75:547–554.
Xia GL, Kikuchi K, Noguchi J, Izaike Y. Short time priming of pig cumulus-oocyte complexes with FSH and forskolin in the presence of hypoxanthine stimulates cumulus cells to secrete a meiosis-activating substance. Theriogenology (2000) 53:1807–1815.[CrossRef][Web of Science][Medline]
Submitted on June 19, 2007; resubmitted on September 19, 2007; accepted on October 1, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Vanhoutte, D. Nogueira, F. Dumortier, and P. De Sutter Assessment of a new in vitro maturation system for mouse and human cumulus-enclosed oocytes: three-dimensional prematuration culture in the presence of a phosphodiesterase 3-inhibitor Hum. Reprod., August 1, 2009; 24(8): 1946 - 1959. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sasseville, F. K. Albuz, N. Cote, C. Guillemette, R. B. Gilchrist, and F. J. Richard Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle Biol Reprod, August 1, 2009; 81(2): 415 - 425. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sasseville, M.-C. Gagnon, C. Guillemette, R. Sullivan, R. B. Gilchrist, and F. J. Richard Regulation of Gap Junctions in Porcine Cumulus-Oocyte Complexes: Contributions of Granulosa Cell Contact, Gonadotropins, and Lipid Rafts Mol. Endocrinol., May 1, 2009; 23(5): 700 - 710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Vanhoutte, D. Nogueira, and P. De Sutter Prematuration of human denuded oocytes in a three-dimensional co-culture system: effects on meiosis progression and developmental competence Hum. Reprod., March 1, 2009; 24(3): 658 - 669. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||