| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Centre de Recherche en Biologie de la Reproduction (C.R.B.R.), Département des Sciences Animales, Université Laval, Ste. Foy, Québec, Canada G1K 7P4
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
cyclic adenosine monophosphate, meiosis, ovum, phosphodiesterases, theca cells
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The somatic components of the follicle have been implicated in the maintenance of meiotic arrest [5–7]. Original studies proposed that the inhibitory factor(s) originated from granulosa cells [8, 9]. However, further experiments showed that isolated theca cell layers or theca cells cultured in monolayers secrete an inhibitory factor capable of maintaining cumulus-oocyte complexes (COCs) in meiotic arrest [7, 10]. Denuded oocytes (DOs) do not respond to the inhibitory action of theca cell monolayers [7, 10]
The second messenger cAMP has been shown to play a key role in the maintenance of bovine oocytes in meiotic arrest. High levels of cAMP in the oocyte induced by the addition of membrane-permeable cAMP derivatives such as dbcAMP or 8-bromo-3',5'-cAMP (8-Br-cAMP) have an inhibitory effect on meiotic resumption [11, 12]. However, high levels of cAMP present in cumulus cells result in meiotic resumption [13, 14]. Therefore, the levels of cAMP present in the somatic or germ cell compartment could be used to modulate the meiotic resumption of bovine oocytes.
The total levels of cAMP found in the oocyte depend primarily on the rate of synthesis by adenylate cyclase (AC) and the rate of degradation by phosphodiesterases (PDE). In the bovine, AC activity has been localized to the cumulus cells and to the plasma membrane of the oocyte [13, 15]. Early studies showed that purines and nucleosides influence the activity of adenylate cyclase [16]. However, it is uncertain whether the oocyte is able to produce enough cAMP to prevent meiotic resumption [17].
Phosphodiesterases hydrolyze the 3' phosphoester bond of the 3',5' purine ribose cyclic monophosphates cAMP and cGMP, thereby transforming them to their corresponding biologically inactive monophosphates. At least 11 different PDE families have been identified since the original purification and characterization of PDE activity [18–22]. Phosphodiesterase families can be identified based on their kinetics and substrate characteristics, inhibitor profiles, allosteric activators and inhibitors, and amino acid sequences [20]. Phosphodiesterases are composed of a catalytic domain, which is connected to the amino and carboxy terminal domains by hinge regions [21]. It is believed that the catalytic domain is structurally homologous between PDEs of the same family, while the other domains act as modulators of the catalytic center [19].
Original experiments used nonselective or first-generation PDE inhibitors, such as IBMX (3-isobutyl-1-methylxanthine), papaverine, theophylline, and pentoxifylline, to study PDE activity [23]. These inhibitors exert no directed action against any one type of PDE but in general inhibit many isozymes. These studies suggested that PDEs are involved in oocyte maturation [24]. The lack of specificity, inhibition of adenosine action, and induction of toxic side effects of these inhibitors limit their usefulness. Approximately 45% of bovine COCs incubated in the presence of 0.2 mM IBMX are temporarily prevented from resuming meiosis [11]. More recently, second-generation or isozyme-specific PDE inhibitors have permitted evaluation of the physiologic role of specific PDE isoenzymes in the overall cyclic nucleotide degradation pathway of the intact cell. These inhibitors act mainly as active site competitors and are used to develop pharmacologic treatments with fewer undesirable side effects [25]. In addition, pharmacologic profiles of individual PDE isoenzymes can be created based on their response to specific PDE inhibitors. The concentration at which these specific inhibitors are used plays a role in their specificity [26–28]. Recent studies used specific PDE inhibitors to demonstrate the presence of PDE3A in rat and mouse oocytes. These inhibitors prevent oocyte maturation both in vivo and in vitro [18, 29].
The PDE3 family shows high affinity for cAMP as a substrate and is inhibited by the presence of cGMP, which binds tightly to the enzyme and it is poorly hydrolyzed. Thus, micromolar concentrations of cGMP greatly inhibit cAMP hydrolysis [30–32]. This PDE family, which is also known as cGMP-inhibited PDE (cGI-PDE), has at least two different gene products: PDE3A and PDE3B. Cilostamide and milrinone are specific PDE3 inhibitors [33].
The PDE4 family or cAMP-specific PDE hydrolyzes cAMP with high affinity. However, the PDE4 family, unlike the PDE3 family, is not inhibited by cGMP [34, 35]. This family has at least four different gene products with two or more alternative splice variants differently expressed in different tissues [20]. Rolipram is routinely used as a specific PDE4 inhibitor.
This study was undertaken to evaluate the specific role of PDE inhibitors on meiotic resumption of bovine oocytes. Since high concentrations of cAMP in the cumulus cells compared to high concentrations of cAMP in the oocyte result in opposite effects on meiotic resumption [13], it is quite possible that these cell types have different PDEs. The focus of this study was placed on the role of type 3 and type 4 PDEs. The effect of type-specific inhibitors was also evaluated in the presence or absence of theca cell monolayers.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bovine theca cell monolayers were prepared as previously described [10]. Bovine follicles measuring 2–5 mm in diameter were randomly chosen from ovaries that were kept on ice in bags containing gauze soaked with saline solution. Follicles were dissected free of stromal tissue with scissors. Theca cells were harvested exclusively from transparent follicles containing at least 75% of the granulosa layer intact and unexpanded COCs. Selected follicles were placed in a calcium- and magnesium-free Hanks balanced salt solution and cut into hemisections with a scalpel. Granulosa cells and COCs were removed by scraping the hemisections with a fine glass loop made from a Pasteur pipette. This technique is used to effectively isolate theca cell layers. The theca layers (n = 20) were enzymatically digested in 15 ml of modified Hanks solution supplemented with 2125 U of collagenase type II, 1600 U trypsin from porcine pancreas, 1 mg DNase type I, and 2 mg EDTA (all from Sigma Chemical Company, St. Louis, MO). Enzymatic digestion took place for 1 h at 38.5°C in an atmosphere of 95% air and 5% CO2. Theca layers were passed through a 10-ml pipette every 20 min. After incubation, larger pieces of remaining follicular hemisections were withdrawn from the solution. The reaction was stopped by the addition of 6 ml of washing medium consisting of Hepes-buffered Tyrode medium (TLH, pH 7.4) [36] supplemented with 10% fetal calf serum (heat-treated FCS; Flow Laboratories, McLean, VA), 0.2 mM pyruvic acid, and 50 µg/ml gentamicin sulfate. Cells were then centrifuged (10 min at 2000 x g), resuspended, and washed a second time. The first and second pellets were resuspended in 10 ml and 1 ml, respectively, of washing medium. Theca cells were counted with a hemocytometer and seeded in a 24-well plate (Falcon Becton Dickinson, Rutherford, NJ) at a concentration of 1.5–2.0 x 105 cells/ml. The culture medium, consisting of TCM-199 with Earle salts (Gibco Laboratories, Grand Island, NY) and bicarbonate (Sigma), 10% FCS, 0.2 mM pyruvic acid, and 50 µg/ml gentamicin sulfate, was pre-exposed to culture conditions (38.5°C, 5% CO2:95% air atmosphere with 100% humidity) for at least 2 h. The culture medium was replenished every 48 h, and follicular cells were incubated for 5–7 days. When the monolayers were confluent, the medium was replenished and conditioned for 48 h.
Collection of COCs
Bovine ovaries at various stages of their reproductive cycle were collected at the slaughterhouse and transported to the laboratory in saline solution kept between 30°C and 35°C. The saline solution consisted of 0.9% NaCl (w:v supplemented with 100 000 IU penicillin, 100 mg streptomycin, and 250 µg amphotericin B per liter, all from Sigma). The contents of follicles measuring 1–5 mm were aspirated with a 10-ml syringe and an 18-gauge needle. The follicular contents were pooled in 50-ml conical tubes. After sedimentation, the COCs were recovered with the use of a stereomicroscope. The COCs used in these experiments had at least five layers of cumulus cells. The COCs were rinsed twice in the supernatant of follicular fluid (centrifuged for 10 min at 350 x g) and then transferred to the respective treatments.
Chemicals
Millimolar stock concentrations of PDE inhibitors were dissolved in dimethyl sulfoxide (DMSO), stored at -20°C, and diluted into culture medium. The chemicals were added to the culture medium or the theca cell monolayers approximately 4 h before the oocytes were added. Final concentrations of DMSO never exceeded 0.1%, and equal amounts of carrier were added to control groups of cells.
Denuded Oocytes
In experiment 1, cumulus cells were removed from the COCs before they were cultured in the presence of PDE inhibitors. Selected COCs were vortexed for 10 min in sterile centrifuge tubes containing follicular fluid supernatant (prepared as indicated previously). After they were vortexed, the oocytes were rinsed in follicular fluid supernatant. Denuded oocytes with a homogeneous cytoplasm were then allocated to their respective treatments.
Fixation of Oocytes
At the end of each treatment, COCs were transferred into 2-ml centrifuge tubes containing 500 µl of TLH and vortex-agitated for 7 min to remove the cumulus cells. Denuded oocytes were recovered under a stereomicroscope and transferred onto a glass slide in a small drop. A Vaseline:paraffin wax mixture was used to maintain the coverslip in contact with the oocytes. The coverslip was fixed in place with epoxy glue, and the slides were immersed in a fixative solution (ethanol:acetic acid, 3:1) for a minimum of 24 h. Oocytes were then stained with 1% aceto-orcein and examined for nuclear morphology with a phase contrast microscope at 100x and 400x magnification [37].
Experimental Design
Experiment 1: effect of PDE inhibitors on the maturation of denuded bovine oocytes In experiment 1, COCs were denuded and then allocated in groups of 15–20 oocytes per treatment. Denuded oocytes were either fixed at time zero or incubated for 12 h in culture medium or culture medium supplemented with the PDE inhibitors cilostamide (10 and 20 µM), milrinone (10 and 50 µM), or rolipram (10 and 50 µM). In addition, a group of oocytes were treated with 0.1% DMSO to determine if the carrier itself used at the highest dose had an effect on meiotic resumption.
Experiment 2: effect of PDE inhibitors on the maturation of bovine COCs In experiment 2, COCs were incubated in culture medium alone or culture medium supplemented with the same PDE inhibitors used in experiment 1. The COCs were denuded after 12 h of treatment and fixed as previously described.
Experiment 3: effect of PDE inhibitors on the maturation of bovine COCs incubated in the presence of theca cell monolayers In experiment 3, groups of 15–20 COCs were incubated for 12 h with untreated theca cell monolayers or theca cell monolayers treated with the same PDE inhibitors used in experiment 2. The effect of DMSO on oocyte maturation was also tested by culturing COCs in the presence of theca cell monolayers supplemented with 0.1% DMSO.
Statistical Analysis
The status of nuclear maturation of 1201 oocytes was evaluated with a contrast microscope immediately after staining with aceto-orcein. Oocytes with a nuclear membrane present were classified as germinal vesicle (GV) stage, while those without a nuclear membrane were classified as having undergone germinal vesicle breakdown (GVBD) and thus meiotic resumption. Results are expressed as mean ± SEM. Data were analyzed by a two-way ANOVA. When ANOVA indicated a significant effect of treatment (P < 0.05), treatment differences were compared by the Duncan multiple range test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
In experiment 2, COCs were incubated in the presence or absence of specific PDE inhibitors for 12 h. Cumulus-oocyte complexes incubated in culture medium resumed meiosis, as did those COCs incubated with the type 4 PDE inhibitor rolipram. In contrast, COCs incubated in the presence of PDE3 inhibitors did not resume meiosis. Cilostamide used at 10 and 20 µM maintained COCs at the GV stage (30.4% ± 15.2% and 46.3% ± 5.3% GV, respectively). Incubation of COCs in the presence of 50 µM milrinone maintained 22.5% ± 0.3% of treated COCs at the GV stage. However, milrinone used at the 10 µM dose maintained only 3.9% ± 2% of those COCs treated in meiotic arrest, which was not significantly different from the percentages of meiotic arrest obtained when COCs were left untreated. Cumulus-oocyte complexes treated with PDE3 inhibitors remain in meiotic arrest, whereas those treated with PDE4 inhibitors resumed meiosis (Fig. 2).
|
In experiment 3, COCs were coincubated with theca cell monolayers in the presence or absence of type-specific PDE inhibitors. Theca cell monolayers maintained 36.5% ± 3.4% of the oocytes in meiotic arrest. The addition of 0.1% DMSO to the coincubated COCs and theca cells did not have a significant effect. The addition of rolipram, a type 4 PDE inhibitor, at either 10 or 50 µM, had no effect on the meiotic arrest maintained by theca cells.
The addition of PDE3 inhibitors to the coincubation significantly increased the number of oocytes arrested at the GV stage (P < 0.05). The percentage of oocytes at the GV stage increased to more than 80% when they were treated with cilostamide (Fig. 3). Milrinone at both 10 and 50 µM also significantly increased the rate of meiotic arrest (61% ± 4.9% and 83.2% ± 7.6% GV, respectively). Under these experimental conditions, cilostamide and milrinone were both equally effective in enhancing the ability of theca cells to maintain the COCs in meiotic arrest.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Raising the levels of cAMP in bovine oocytes by treating them with an activator of AC (forskolin) or cAMP analogues (dbcAMP and 8-Br-cAMP) transiently delays meiotic resumption, thus supporting the idea that high levels of cAMP in the oocyte affect oocyte maturation [14]. Electron microscopy studies have shown that an increase in AC levels in the cumulus cell projections contacting the oocyte occurs only after forskolin stimulation. In the absence of this stimulation, the oocyte might not have enough AC enzymatic activity to affect its nuclear maturation [15].
Preventing the degradation of cAMP by means of treatment with broad-spectrum PDE inhibitors such as IBMX can also transiently delay meiotic resumption [11]. A transient effect of IBMX on meiotic arrest was observed after incubating bovine COCs with IBMX for 8 h; however, this effect was not noticeable after 24 h [13]. In addition, IBMX is more effective in preventing the resumption of meiosis in zona-free oocytes than in COCs. Treatment of COCs with IBMX increases the levels of cAMP in both the cumulus cells and the oocyte. Cumulus-oocyte complexes treated with IBMX contain approximately twice the level of cAMP found in untreated COCs [13].
In this study, PDE3 and PDE4 inhibitors were used to determine whether type 3 and type 4 PDEs play a role in modulating the resumption of meiosis of bovine oocytes. The PDE3 inhibitors cilostamide and milrinone maintained bovine oocytes in meiotic arrest. On the other hand, the PDE4 inhibitor rolipram used under the same experimental conditions did not block oocyte maturation. These results suggest a role for PDE3 in the meiotic arrest of bovine COCs and more specifically in denuded bovine oocytes. The ability of cilostamide and milrinone to inhibit meiotic resumption suggests that inhibition of PDE3 prevents meiotic resumption in bovine oocytes [41]. Similar results have been observed in rat oocytes both in vitro [29] and in vivo [42]. Cilostamide is more effective than milrinone in maintaining meiotic arrest in bovine oocytes. The higher dose required to obtain meiotic arrest with milrinone has also been reported in rat oocytes [29].
Theca cell monolayers have been shown to be effective in maintaining bovine COCs, but not DOs, in meiotic arrest [10, 43]. Our laboratory has demonstrated that the inhibitory factor produced by the theca cell requires that the cumulus cells be in direct contact with the oocyte. The cumulus cells play a key role in either transporting or processing the inhibitory factor produced by the theca cell monolayers [10]. In addition, modulation of cAMP is important in controlling meiotic arrest of bovine COCs incubated with theca cell monolayers [43]. The addition of PDE3 inhibitors to the coincubation of COCs and theca cell monolayers had an additive effect on meiotic arrest. Incubation of COCs in the presence of theca cell monolayers and type 3 PDE inhibitors produced higher rates of meiotic arrest than those obtained by incubating the COCs in the presence of untreated theca cell monolayers. However, the type 4 PDE inhibitor rolipram did not have an additive effect. The additive effect observed by treating theca cell monolayers with PDE3 inhibitors suggests that the factor(s) produced by the theca cell monolayers act upstream from the site of action of PDE3 inhibition. The theca cell factor(s) may act indirectly by decreasing the activity of type 3 PDEs. We suggest that these two inhibitory mechanisms are complementary or possibly alternative methods to maintain meiotic arrest. Phosphodiesterase inhibitors will act by regulating cAMP, whereas theca cells might use one or more alternative mechanisms. Unfortunately, the mechanism(s) used by theca cell monolayers to maintain meiotic arrest has not yet been elucidated.
Another way to explain these results is based on the ability of the COCs to respond to the inhibitory signals. There might be differences in the oocytes' ability to respond to the inhibitory signals despite the care taken to select, as much as possible, a morphologically homogenous population of COCs. This hypothesis is supported by the fact that neither system is capable of maintaining all of the treated oocytes in meiotic arrest. When used in conjunction, both systems might be able to target more oocytes than either one alone. The remaining unresponsive oocytes are probably already committed to meiotic resumption.
Incubation of oocytes with rolipram had no significant effect under any other of the experimental conditions tested. This finding suggests that PDE4 is not involved in the maintenance of meiotic arrest of bovine oocytes. Because of the considerable amount of research done both in vitro [25, 29, 44] and in vivo with rolipram [42] we believe that the product's inability to maintain meiotic arrest is not a result of a lack of permeability across the plasma membrane.
Differential regulation of cAMP levels by PDEs might explain the role of cAMP in meiotic resumption. The localization of active PDE subtypes to different cell type compartments such as somatic or germ cells appears to play an important role in the regulation of meiosis [29, 38]. It was recently reported that treatment of mural granulosa cells with the PDE4 inhibitor rolipram resulted in a 2.5-fold increase in the level of cAMP relative to controls, whereas treatment with the PDE3 inhibitor milrinone did not increase cAMP levels in those cells [45]. Therefore, preferential activation of certain PDE subtypes in the cumulus cells or the oocyte might determine whether the oocyte remains in meiotic arrest or proceeds to meiotic resumption.
In conclusion, the results of this study show that inhibition of PDE3 prevents meiotic resumption of bovine oocytes, whereas inhibition of PDE4 does not block oocyte maturation. Furthermore, the additive effect of PDE3 inhibitors on the meiotic arrest maintained by theca cell monolayers suggests that PDE3 plays a role in meiotic maturation of bovine oocytes.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). 
2 Correspondence: Marc-André Sirard, Département des Sciences Animales, Pavillon Paul-Comtois, Cité Universitaire, Ste. Foy, PQ, Canada G1K 7P4. FAX: 418 656 3766; marc-andre.sirard{at}crbr.ulaval.ca
Accepted: August 28, 2001.
Received: January 31, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. A. Mayes, M. F. Laforest, C. Guillemette, R. B. Gilchrist, and F. J. Richard Adenosine 5'-Monophosphate Kinase-Activated Protein Kinase (PRKA) Activators Delay Meiotic Resumption in Porcine Oocytes Biol Reprod, April 1, 2007; 76(4): 589 - 597. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. J. Richard Regulation of meiotic maturation J Anim Sci, March 1, 2007; 85(13_suppl): E4 - E6. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Nogueira, R. Ron-El, S. Friedler, M. Schachter, A. Raziel, R. Cortvrindt, and J. Smitz Meiotic Arrest In Vitro by Phosphodiesterase 3-Inhibitor Enhances Maturation Capacity of Human Oocytes and Allows Subsequent Embryonic Development Biol Reprod, January 1, 2006; 74(1): 177 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Pace and P. Thomas Steroid-Induced Oocyte Maturation in Atlantic Croaker (Micropogonias undulatus) Is Dependent on Activation of the Phosphatidylinositol 3-Kinase/Akt Signal Transduction Pathway Biol Reprod, November 1, 2005; 73(5): 988 - 996. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D Nogueira, R Cortvrindt, B Everaerdt, and J Smitz Effects of long-term in vitro exposure to phosphodiesterase type-3 inhibitors on follicle and oocyte development Reproduction, August 1, 2005; 130(2): 177 - 186. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R.E. Thomas, J.G. Thompson, D.T. Armstrong, and R.B. Gilchrist Effect of Specific Phosphodiesterase Isoenzyme Inhibitors During In Vitro Maturation of Bovine Oocytes on Meiotic and Developmental Capacity Biol Reprod, October 1, 2004; 71(4): 1142 - 1149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Thomas, D. T. Armstrong, and R. B. Gilchrist 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, March 1, 2004; 70(3): 548 - 556. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Luciano, S. Modina, R. Vassena, E. Milanesi, A. Lauria, and F. Gandolfi 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, February 1, 2004; 70(2): 465 - 472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Nogueira, R. Cortvrindt, D.G. De Matos, L. Vanhoutte, and J. Smitz Effect of Phosphodiesterase Type 3 Inhibitor on Developmental Competence of Immature Mouse Oocytes In Vitro Biol Reprod, December 1, 2003; 69(6): 2045 - 2052. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Nogueira, C. Albano, T. Adriaenssens, R. Cortvrindt, C. Bourgain, P. Devroey, and J. Smitz Human Oocytes Reversibly Arrested in Prophase I by Phosphodiesterase Type 3 Inhibitor In Vitro Biol Reprod, September 1, 2003; 69(3): 1042 - 1052. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
