| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gamete Biology |
Institute of Anatomy of Domestic Animals, Histology and Embryology, Faculty of Veterinary Medicine, University of Milan, Milan, 20133, Italy
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
cumulus cells, cyclic adenosine monophosphate, gamete biology, oocyte development
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Intercellular communications within the ovarian follicle have been implicated in the control of meiotic arrest and maturation of mammalian oocytes, and such a metabolic coupling in cumulus-oocyte complexes (COCs) before the beginning of maturation has been demonstrated to be essential in the acquisition of developmental competence [10].
During gonadotropin-induced ovulation at midcycle, the number of GJs decreases in parallel with the meiotic resumption of the oocyte [5, 11, 12]. At that time this event is accompanied by the progressive expansion of cumulus oophorus and hyaluronic acid synthesis. Both of them are induced in vitro by FSH [13] via a mechanism that appears to be mediated by cAMP both in rat and cow [14, 15]. Cumulus cell expansion in vitro correlates with hyaluronic acid synthesis starting at 3 h from the beginning of maturation to 18 h of incubation with FSH; in the absence of the hormone, only 10% of hyaluronic acid is produced, and COC expansion does not occur [16].
FSH and LH exert their function by activating membrane receptors leading to the production of cAMP through adenylate cyclase. Cyclic AMP acts as the intracellular messenger for gonadotropin stimulation [17, 18]. However, the precise mechanism by which intracellular concentration of cAMP ([cAMP]i) evokes a stimulatory or inhibitory response by the oocyte during meiotic events is not fully understood. High levels of cAMP have been proposed as the regulatory mechanism to maintain the oocyte in meiotic arrest. In fact, when oocytes are removed from the follicular compartment and cultured with agents that maintain high intracellular concentrations of cAMP, they remain in the germinal vesicle (GV) stage. Moreover, membrane-permeable cAMP analogues (e.g., dibutyryl cAMP, 8-Br-cAMP); forskolin, which activates adenylate cyclase or the nonselective phosphodiesterase inhibitor; and 3-isobutyl-1-methylxanthine (IBMX), which prevents cAMP degradation, are all able to inhibit spontaneous oocyte maturation [19–22]. However, transient elevation of cAMP can induced oocyte maturation in vitro by cAMP analogues or IBMX [23, 24]. Consequently, the stimulatory or inhibitory effect of cAMP is presumably dependent on the levels of cAMP in different compartments of the follicle.
We previously reported that bovine oocytes can be matured in vitro in defined medium containing invasive adenylate cyclase (iAC), an enzyme purified from Bordetella pertussis that can modulate the intracellular concentration of cAMP [25]. High concentrations of iAC can reversibly inhibit meiotic progression [26], whereas low concentrations can promote oocyte maturation and development after fertilization [25]. In vitro maturation in the presence of 0.01 µg/ml of iAC resulted in a blastocyst rate that was significantly greater than that obtained after in vitro maturation (IVM) in the absence of stimulation or standard maturation medium supplemented with fetal bovine serum (FCS) and gonadotropins. Interestingly, iAC-mediated maturation was not accompanied by cumulus expansion.
Based on these findings, the present study was designed to investigate the physiological role played by cAMP induced by maturation conditions known to promote different levels of cumulus expansion as well as the achievement of different developmental competence. In accord with our previous study [25], COCs were matured alternatively 1) in the absence of stimulation (low competence), 2) with serum and gonadotropins (standard competence), or 3) with iAC (high competence). During in vitro maturation, [cAMP]i was determined in both COC and oocyte, and the dynamic changes of GJ-mediated communications between oocyte and cumulus cells were analyzed by the dye coupling technique and immunohistochemical localization of Cx43.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Oocyte Collection, Selection, and In Vitro Maturation
Bovine ovaries were obtained from a local abattoir and transported to the laboratory, within 2 h, in Dulbecco PBS maintained at 32°C–34°C. As previously described, only ovaries with more than 10 follicles were processed for oocyte aspiration [27]. All subsequent procedures were conducted at a constant temperature of 36°C.
COC isolation and selection were performed in basic collection media (bCM) or complete collection media (cCM). Basic CM was TCM-199 supplemented with 0.1% polyvinyl alcohol (PVA), 25 mM Hepes, and 10 µg/ml heparin. Complete CM was made with bCM supplemented with 0.5 mM 3-IBMX and 0.1 µg/ml iAC [25]. The IBMX and iAC were included in order to preserve the cAMP concentration in COCs as closely as possible to that present when they were within the follicle, as suggested by Aktas et al. [28].
The COCs were retrieved from the ovaries by aspiration of 2–6 mm follicles with an 18-gauge needle on a 10 ml syringe containing a small volume of collection medium. The final proportion of follicular fluid to collection medium was maintained as closely as possible to 3:1. The COCs were examined under a stereomicroscope, and only those medium-brown in color with three or more complete layers of cumulus cells and a finely granulated homogenous ooplasm were used. Selected COCs were then washed two times in the same medium used for collection and two times in the maturation medium used according to the experimental treatments described in Table 1. The whole procedure was performed in approximately 30 min.
|
The basic maturation medium was TCM-199 supplemented with 0.68 mM L-glutamine, 25 mM NaHCO3. To this was added 0.1% of PVA (control) or 10% (v/v) fetal bovine serum plus 0.1 IU/ml human menopausal gonadotropins (hMG; Pergovet Serono, Rome, Italy; FCS+hMG) or 0.1% of PVA and 0.01 µg/ml iAC (iAC). The COCs were cultured in 500 µl of maturation medium for 24 h in four-well dishes (Nunc-VWR International, Milan, Italy) at 38.5°C under 5% CO2 in humidified air.
Oocyte In Vitro Fertilization and Embryo Culture
After maturation, cumulus expansion was evaluated in all treatments before fertilization. Groups of COCs of the three maturation conditions were fertilized, and embryos were cultured as previously described [25]. Briefly, the contents of a straw of cryopreserved bull spermatozoa (CIZ, S. Miniato, Italy) was thawed and cells separated on a 45%–90% Percoll gradient. Sperm was counted and diluted to a final concentration of 1 x 106 spermatozoa per milliliter of fertilization medium, which was a modified Tyrode solution [29] supplemented with 0.6% (w/v) BSA fatty acid free, 10 µg/ml heparin, 20 µM penicillamine, 1 µM epinephrine, and 100 µM hypotaurine. Groups of 25–30 COCs were added to 300 µl of fertilization medium and incubated for 18–20 h in four-well dishes at 38.5°C under 5% CO2 in humidified air.
After fertilization, presumptive zygotes were denuded by vortexing for 3 min in 500 µl of Tyrode solution (TL-Hepes [29]) and then transferred in groups of 20–30 to the culture drops of 20 µl made with M199 supplemented with 10% of FCS in the presence of bovine oviduct epithelial cells. Incubation was performed under mineral oil at 38.5°C under 5% CO2 in humidified air. Cleaved eggs were transferred into a fresh drop 48 h postinsemination. Thereafter, 20 µl of fresh medium were added every 2 days to each drop and then an equal amount was removed to maintain a constant volume. Blastocyst rate was assessed at 186 h postinsemination, and embryo cell nuclei were counted with propidium iodide.
Determination of Intracellular Concentration of cAMP in COC and Oocytes
In order to determine the content of cAMP in single COC or oocytes, groups of 30–40 COCs were cultured as described above. At time intervals of 0, 3, 7, 12, 18, and 24 h from the beginning of maturation, COCs were harvested and [cAMP]i was determined in groups of 5–10 whole COCs or in groups of 10–20 oocytes obtained after cumulus cells removal before assay. Oocytes were denuded by gently pipetting in TL-Hepes containing 0.5 mM of IBMX. The complete removal of cumulus cells from oocytes was monitored using a stereomicroscope. Denuded oocytes were washed two times in the same buffer containing IBMX to remove dispersed cumulus cells. Finally, intact COCs and denuded oocytes were washed two times in fresh TL-Hepes in the absence of IBMX, transferred in a minimum volume of washing buffer ranging between 3 and 5 µl in an Eppendorf tube, and snap frozen in liquid nitrogen and stored at -20°C until assayed. The same procedures were applied to selected COCs after follicle removal (5 min from aspiration) and after aspiration and selection procedures with different collection media 30 min later at the beginning of culture (time 0). Intracellular concentration of cAMP was determined by a competitive enzyme immunoassay system (EIA, Biotrak, Amersham Life Science, Milan, Italy) with acetylation protocol for highest test sensitivity according to the procedure provided with the kit. At the end of the procedures, optical density of samples was determined in a plate reader at 450 nm within 30 min. Each sample was tested in triplicate in each experiment. The assay was validated by adding increasing numbers of COCs or oocytes (20, 50, and 100) to a constant volume of extraction medium and recovering a proportional amount of cAMP.
Dye Coupling Experiment
Intercellular communication between oocyte and cumulus cells was assessed by a Lucifer yellow dye (LY) microinjection. Groups of 30–40 cumulus-oocyte complexes were incubated in maturation media as above described for 0, 3, 7, 12, 18, and 24 h. At each time interval, a 3% solution of LY in 5 mM of lithium chloride was pressure injected into the oocyte, and the spread of dye into surrounding cumulus cells was monitored with an inverted fluorescence microscope (Nikon Diaphot; Nikon Corp., Tokyo, Japan). A Narishige microinjection apparatus (Narishige CO LTD, Tokyo, Japan) was used to guide the holding and injecting micropipettes into a 50 µl drop of TL-Hepes covered with mineral oil. A total of 556 COCs were injected at different intervals of time during in vitro culture. Analysis of GJ functionality was performed within 10 min after injection by observation of LY spreading from oocyte to cumulus cells [30]. GJ-mediated oocyte-cumulus cell communications were classified as open, partially open, and closed. COCs were classified partially open when only a limited number of cells showed signs of dye diffusion between ooplasm and corona radiata cells.
Immunohistochemical Localization of Cx43
The morphological study of GJ was performed by indirect immunofluorescence localization of Cx43. In each experiment, 10 COCs for each group of maturation time were fixed as "whole mount" in 2.5% paraformaldehyde (w/v) in PBS and processed following the protocol suggested by Sutovsky et al. [8]. An anti-Cx43 monoclonal antibody diluted in 1:350 PBS (Zymed Laboratories, San Francisco, CA) was used as the primary antibody, and an FITC-conjugated anti-mouse IgG diluted 1:300 in PBS (Jackson Immunoresearch Lab, West Grove, PA) was used as a secondary antibody. COCs were examined with a Confocal Laser Scannig Microscope (Bio-Rad Laboratories, Hercules, CA). Each analysis at each time point was replicated three times on different batches of COCs.
Statistical Analysis
All experiments were replicated at least three times. The data are expressed as mean ± SEM. The data of in vitro embryo development were analyzed using one-way ANOVA followed by Duncan New Multiple Range test (SuperANOVA, Abacus Concepts, CA). Differences between COC or oocyte cAMP measurements and dye transfer experiment were determined after logarithmic transformation of data using one-way ANOVA, and differences between treatment means were tested using Student-Newman-Keuls post hoc comparison. Probabilities of less than 0.05 were considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
As summarized in Table 2, only COCs cultured in the presence of serum and gonadotropins showed a complete cumulus expansion after 24 h of IVM, whereas control and iAC 0.01 groups did not. The absence of stimulation in control medium resulted in the lowest cleavage and developmental rates compared with the other groups. The administration of 0.01 µg of iAC during IVM induced a cleavage rate similar to the FCS+hMG group, but a blastocyst rate significantly higher than the other maturation conditions. No differences of blastocyst cell number were observed between all treatments.
|
Intracellular Concentration of cAMP in COC and Oocyte
The supplementation of iAC and IBMX to the collection and selection medium prevented a drop of intracellular cAMP during the COCs collection procedures, both in COC and in the oocytes. As shown in Table 3, shortly after the isolation from the follicle (5 min of procedures), COCs collected with bCM had a COC intracellular concentration of cAMP similar to those collected with cCM. However, after 30 min of manipulation procedures (Time 0), only COCs isolated in cCM had a [cAMP]i similar to the amount they possessed after removal from the follicle, whereas a significant drop in [cAMP]i occurred when COCs were collected with bCM. Oocyte cAMP content immediately after isolation from the follicle was similar for both collection media used, whereas a drop occurred after 30 min of manipulation procedures (Time 0) when COCs were isolated in the presence of bCM.
|
After 3 h of culture, intracellular concentration of cAMP in COC increased in all three groups compared with Time 0. This increase was significantly higher in both FCS+hMG and iAC groups than control during all the maturation times (Table 4). Moreover, between 3 and 7 h the FCS+hMG group showed a peak, significantly higher than iAC treatment and temporally correlated to the beginning of cumulus expansion process, which took place only in this group.
|
The administration of iAC or FCS+hMG during in vitro maturation elicited a moderate increase in oocyte cAMP content after 3 h of culture, similar in both treatments but significantly higher than control (Table 5). However, in the FCS+hMG treatment, intraoocyte cAMP concentration decreased from starting from 7 h up to 18 h of maturation to a level similar to the control group. In the iAC group, the cAMP concentration in oocytes was significantly higher than FCS+hMG and control groups through the entire maturation period.
|
Dye Coupling Experiment
Injection of LY in the oocytes at Time 0 resulted in an immediate spread of the dye into neighboring corona radiata cells in 69.3% ± 7.2% of COCs, whereas 19.1% ± 4.8% and 11.5% ± 2.6% of COCs showed a pattern of partially and completely closed communications, respectively (Fig. 1). At this time, no differences were observed in the above proportion, despite the collection media used during COC isolation.
|
A dramatic drop in oocyte-cumulus cell coupling occurred in the control group starting from 3 h of culture (Table 6), where only 34.8% of COCs exhibited open intercellular junctions, a percentage significantly lower than the other groups. In the FCS+hMG group, the presence of functional GJ-mediated communications between oocytes and surrounding cumulus cells were observed up to 7 h from the beginning of maturation in 43.5% of COCs, whereas they declined to 5.5% after 12 h of culture. COCs matured in the presence of iAC resulted in open communications for up to 12 h of IVM in 47.7% of COCs (Table 6). Starting from 18 h to the end of IVM, no dye diffusion was observed between oocyte and cumulus cells in any group.
|
Immunohistochemical Localization of Cx43
At Time 0 of IVM, immunohistochemical localization of Cx43 showed no signal visible in the cytoplasm in all oocytes examined (Fig. 2A), whereas an occasional weak positive signal crossing zona pellucida was detected (Fig. 2C, arrow). In cumulus cells, Cx43 was organized in large bright dots or dashes at the cell-to-cell contact sites (Fig. 2F).
|
After 3 h of IVM in the control group, 7 h in the FCS+hMG group, and 12 h in the iAC group, Cx43-positive pleomorphic spots appeared inside the cytoplasm of the oocyte (Fig. 2B). Cumulus cells showed GJs organized in plaques, as at Time 0; however, a positive staining was localized also inside the cytoplasm of the cells (Fig. 2G). After 7 h of IVM in the control group and after 12 h in the FCS+hMG group, Cx43-positive large vesicles localized inside the cytoplasm of the oocytes became visible (Fig. 2D). GJ among cumulus cells and between cumulus cells and oocytes evolved in the same manner as those in COCs matured in the presence of iAC. However, Cx43-positive large vesicles localized inside the cytoplasm of the oocytes (Fig. 2D) became detectable only after 18 h of maturation, later than in control and FCS+hMG groups.
After 24 h of IVM in all of the groups, oocytes showed a diffuse positive signal inside the cytoplasm (Fig. 2E), whereas small punctuate plaques were present only between the few cumulus cells still in contact.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The results of this study indicate that the presence of IBMX and iAC from the time of COC isolation is able to maintain the original intracellular concentration of cAMP retained by the whole COC and the oocyte immediately after removal from the follicle until the beginning of maturation. Moreover, oocyte maturation and cumulus expansion are triggered by two different intracellular levels of cAMP, such that the cAMP level required to stimulate meiotic resumption is lower than that required for cumulus expansion. Intracellular cAMP concentrations induced in cumulus cells and oocytes are different in the three IVM media used. It is interesting to note that different concentrations of cAMP correspond to different dynamic changes of coupling between oocyte and cumulus cells. The highest rates of oocyte maturation and developmental competence are accompanied by the persistence of cumulus-oocyte communications. Intercellular communications are normally interrupted following cumulus expansion; they cease, however, with a certain delay when expansion does not occur. In our experiment, in absence of stimulation, cAMP content in both COCs and oocytes remained significantly lower than the other two groups, and intercellular communications declined dramatically during the culture period, prior to the other treatments. The presence of serum and gonadotropins in the maturation medium stimulated a sharp intracellular cAMP increase between 3 and 7 h of maturation in COCs, followed by a decline to a level higher than the control group up to the end of maturation. This peak could be related to the beginning of the cumulus expansion process, which takes place only in this treatment.
Similar to the FCS+hMG group, COCs treated with 0.01 µg/ml of iAC showed an intraocyte [cAMP]i peak at 3 h, significantly higher than in control but lower than in the FCS+hMG group, followed by a leveling off of [cAMP]i during the entire maturation period, similar to COCs stimulated with FCS+hMG. However, the [cAMP]i increase in the FCS+hMG group is followed by a significant decrease in cell coupling, whereas the weak increase in the iAC treatment is associated with a delay of the interruption of intracellular communications in a significant proportion of the COCs analyzed.
In oocytes, both the FCS+hMG and iAC groups showed a similar peak at 3 h, significantly higher than in the control. Starting from 7 h of culture, the oocyte cAMP content in COCs stimulated with iAC was significantly higher than the other groups until the end of the maturation period. These data suggest that in cattle, [cAMP]i required for meiotic resumption and developmental competence is lower than that required for cumulus expansion. In addition, below a threshold level oocyte maturation becomes defective, leading to a low developmental competence.
It is interesting to note that iAC treatment does not contain serum; therefore the effect of unknown factor(s) present in the FCS+hMG group can be hypothesized.
The participation of cyclic nucleotide cAMP in the control of oocyte maturation in mammals has been indicated in the past years. Nevertheless, the molecular mechanisms and the series of coordinate events guided by cAMP have not been clearly defined. Our data are consistent with [cAMP]i in oocytes and whole COCs previously measured in cows at the time of follicle removal [28, 31]. In particular, in our experiments the intracellular cAMP concentration during the interval between oocyte isolation from the follicle and the beginning of in vitro maturation seems critical for the achievement of higher developmental competence. The use of compounds, which prevent a decrease of [cAMP]i before IVM, can be useful from the time COCs are isolated from the follicles [22, 25, 26, 28]. This is in agreement with previous findings, where the simple maintenance of intracellular cAMP during COC isolation induces a higher developmental rate than COCs isolated in simple medium [25, 32].
An intriguing question is how the exogenous modulation of cAMP can evoke a stimulatory or inhibitory response in germinal and somatic compartments. High levels of cAMP inhibit spontaneous oocyte maturation [33, 34]. This can be achieved by using analogs of cAMP or phosphodiesterase inhibitors that prevent cAMP intracellular degradation or adenylate cyclase activating agents. In our experiments, intraoocyte cAMP concentration induced by 0.01 µg/ml of iAC between 3 and 7 h of maturation was similar to that induced by serum and gonadotropin, whereas the COC intracellular concentration of cAMP that stimulated cumulus expansion was several times higher in FCS+hMG stimulation.
A possible explanation of these apparently opposing effects may be the compartmentalization and the differential activity of phosphodiesterases (PDEs) in the oocyte and cumulus cells. Recent studies have demonstrated the presence of oocyte and cumulus cell-specific PDE isoenzymes [35–37]. The selective regulation and expression of PDEs may account for the apparently paradoxical role of cAMP and may be involved in the modulation of cAMP levels in oocyte and cumulus cell compartments independently during maturation. The differential activity of the two isoenzymes within the oocyte and the cumulus cells may be responsible for the response of oocyte and cumulus cells to cAMP. Our study further supports earlier studies proposing that the relative changes in cAMP levels could be more responsible for meiotic resumption than the absolute concentration of cAMP in the oocyte [38, 39].
Oocyte-cumulus cell coupling is regulated by dynamic changes in Cx43 GJ-mediated communications as previously reported in pigs and cows [8, 40]. Our data show that at Time 0 in all groups and after 3 h of IVM in both FCS+hMG and iAC 0.01 groups, no evident signal for Cx43 was visible between cumulus cells in contact with the oocyte. Since the presence of functional GJ at this time has been previously demonstrated [8], we suggest that GJ between corona radiata cells and oocytes have reduced size. In fact, it has been demonstrated that functional GJs are arranged into plaques that can extend from several nanometers to several micrometers in diameter [41]; however, plaques of less than 0.1 µm are not visible by immunofluorescence microscopy [42]. At Time 0 we have observed a weak positive signal for Cx43 crossing zona pellucida. This indicates the presence of junctions localized between transzonal projections originating from cumulus cells that contact the oocyte surface [6]. Many of the subunit proteins that form connexons are rather dynamic, with a half life of only a few hours [43]. Degradation of GJ requires the removal of older connexins from the center of the junctions and the internalization within intracellular vesicles of different sizes [44]. In the present study, we observed that the interruption of communications is associated with the appearance of pleomorphic vesicles inside the cytoplasm of the oocyte. We interpret these particular structures as endocytotic vesicles containing Cx43 derived from internalization and destruction of the corona radiata-oocyte specific GJ.
Disappearance of functional GJ after 7 h of IVM in the FCS+hMG group is also in agreement with data presented by Sutovsky et al. [8]. In the present study, iAC treatment shows a delay of 5 h in both cell-oocyte coupling interruption and the subsequent Cx43 turnover. This coincides with the persistence of GJ-mediated communications observed up to 12 h in COCs matured in the presence of iAC.
In the cumulus cells, the presence of positive large plaques between cells in all of the treatments indicated that there also is a direct communication between contacting cells during IVM, which is preserved even at the end of maturation time when GJs are organized in punctuate plaques. The diffuse signal inside the cytoplasm may indicate a turnover of the connexons, as previously described in other somatic cells [45].
The present study brings further elements in understanding the series of events leading to the final maturation of bovine oocytes. Both functional and morphological analysis of GJs between oocytes and cumulus cells indicated that high maturation and development rates are accompanied by the persistence of permeable communications, which, however, are independent from cumulus expansion. On the contrary, the premature interruption of such communications was linked to low maturation and development capabilities. As previously demonstrated, the junctional pathways disruption is not essential to start meiotic resumption, but intercellular and metabolic coupling is determinant for developmental competence [10]. The effect of a low concentration of cAMP seems to promote a prolongation of the cross-talk interaction between somatic and germinal compartments resulting in a higher developmental capacity after fertilization. This supports previous works aimed to promote a higher developmental potential by prematuration treatments in reversible GV-arrested bovine oocytes [32, 46, 47].
In conclusion, intracellular levels of cAMP have a profound effect on oocyte developmental competence starting from COC removal from the follicle to the end of maturation. A tuned modulation of intracellular cAMP levels within a physiological range in the oocyte and cumulus cells during in vitro maturation lead to the acquisition of high developmental potential after fertilization. We hypothesize that the higher developmental potential of COCs matured in the presence of a low concentration of iAC could be due to a moderate increase of oocyte intracellular cAMP, which in turn could exert its positive effects on developmental competence by delaying the natural interruption of oocyte-cumulus cells communications.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Alberto M. Luciano, Istituto di Anatomia degli Animali Domestici con Istologia ed Embriologia, Università degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy. FAX: 39 02 5031 7980; alberto.luciano{at}unimi.it
Received: 26 June 2003.
First decision: 17 July 2003.
Accepted: 24 September 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  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, 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., April 23, 2009; (2009) dep104v1. [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]
|
|
|
|
|
|
Y.-m. Shu, H.-t. Zeng, Z. Ren, G.-l. Zhuang, X.-y. Liang, H.-w. Shen, S.-z. Yao, P.-q. Ke, and N.-n. Wang Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes Hum. Reprod., March 1, 2008; 23(3): 504 - 513. [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]
|
|
|
|
 |
|
 D. Pant, L. P Reynolds, J. S Luther, P. P Borowicz, T. M Stenbak, J. J Bilski, R. M Weigl, F. Lopes, K. Petry, M. L. Johnson, et al. Expression of connexin 43 and gap junctional intercellular communication in the cumulus-oocyte complex in sheep Reproduction, February 1, 2005; 129(2): 191 - 200. [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]
|
|
|
|
|
|
M. L Sutton-McDowall, R. B Gilchrist, and J. G Thompson Cumulus expansion and glucose utilisation by bovine cumulus-oocyte complexes during in vitro maturation: the influence of glucosamine and follicle-stimulating hormone Reproduction, September 1, 2004; 128(3): 313 - 319. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
