1 TECNOBIOS Procreazione, Bologna, 40125, 2 Department of Biomedical Sciences and Technologies, 3 Department of Experimental Medicine, University of L'Aquila, L'Aquila, 67010, Italy, 4 Novo Nordisk A/S, Copenhagen, 2820, Denmark, 5 University of Bologna, 40125, Bologna, Italy and 6 Department of Obstetrics & Gynaecology, University of Sydney, Sydney, NSW 2145, Australia
7 To whom correspondence should be addressed at: Department of Sciences and Biomedical Technologies, University of L'Aquila, Via Vetoio, Loc. Coppito, 67010 Coppito, L'Aquila, Italy. Email: cecconi{at}univaq.it
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Abstract |
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Key words: cumulus cells/FSH/meiosis/oocyte development
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Introduction |
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At the end of the follicular phase, meiotic maturation is finally promoted by a stimulus triggered by the midcycle LH surge. The chain of events leading to germinal vesicle breakdown (GVBD) and PBI emission is only partially known, despite extensive studies conducted especially in the mouse. It is possible that, after acute LH stimulation, the inhibitory influence of the follicular environment is withdrawn. This could occur as a consequence of the breakdown in gap junction communication between the cumulus mass and the rest of the follicle (Dekel et al., 1988), leading to the interruption of the transfer of inhibitory molecules to the oocyte. But a role for a positive stimulus has also been suggested. This is inferred by the observation that in mouse cumulus-enclosed oocytes (CEO), meiotic resumption in vivo (Su et al., 2002
) and in vitro under conditions which maintain continued meiotic arrest (Downs et al., 1988
; Su et al., 2002
, 2003
) can be induced by an overriding signal originating in cumulus cells as a direct effect of gonadotrophin action.
The participation of the somatic compartment appears fundamental for gonadotrophic action, since pre-ovulatory oocytes devoid of cumulus cellsnaked oocytes (NkO)are insensitive to FSH. In addition, it has been reported that intact oocytecumulus cell gap junctions are essential in order to deliver to the oocyte the inductive message prompted by FSH (Fagbohun and Downs, 1991). However, the involvement of paracrine signals has also been proposed. Indeed, a small group of intermediates of cholesterol biosynthesis may represent at least part of the physiological signal that originates in the somatic compartment of the follicle and instructs the oocyte to reinitiate the meiotic cycle. In particular, human follicular fluid has been found to contain an activity, identified as 4,4-dimethyl-5
-cholest-8,14,24-trien-3
-ol, known as follicular fluid meiosis-activating sterol (FF-MAS) (Byskov et al., 1995
). This agent promotes maturation in germinal vesicle (GV) stage mouse oocytes maintained in meiotic arrest with Hx, either in the presence or absence of surrounding cumulus cells (Grøndahl et al., 1998
). Irrespective of how stimulated cumulus cells transmit the inductive stimulus, regulation of intra-oocyte cAMP degradation is a key element in meiotic control. This is supported by the finding that selective inhibition of the oocyte-specific PDE 3 with cilostamide causes GV arrest either in vivo in the mouse (Wiersma et al., 1998
) or in vitro in the mouse (Tsafriri et al., 1996
), bovine (Thomas et al., 2002
) and human (Nogueira et al., 2003
) oocytes.
On the basis of these findings, the aim of our study was to evaluate: (i) the relative influence of FSH, EGF and FF-MAS on meiotic maturation of mouse CEO maintained in the continued presence of different meiotic inhibitors, such as Hx, dbcAMP and cilostamide; and (ii) the hypothesis that the action of FSH and EGF may be mediated by a cumulus cell-derived paracrine signal.
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Materials and methods |
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Oocyte isolation
Three week old immature Swiss CD1 female mice (Harlan, Italy) were primed with 5 IU pregnant mare serum gonadotrophin (Folligon; Intervet, Italy) and killed 48 h later. Intact CEO were released by puncturing large antral follicles. The medium used for oocyte collection was either HEPES-buffered -minimum essential medium (
-MEM; Invitrogen Corporation, UK) supplemented with antibiotics (100 IU/ml penicillin and 50 µg/ml streptomycin), pyruvate (0.23 mmol/l), glutamine (2 mmol/l) and bovine serum albumin (BSA) (8 mg/ml), or HEPES-buffered Cook Oocyte Wash (Cook IVF, Australia). To prevent spontaneous resumption of meiosis, the collection medium was supplemented with either 0.3 mmol/l dbcAMP, 8.5 µmol/l cilostamide or 4 mmol/l Hx, depending on subsequent in vitro maturation conditions.
Assessment of meiotic maturation in vitro
Groups of 3040 CEO were transferred into 6080 µl of in vitro maturation medium (1 CEO/2 µl of medium) in 96-well plates. The IVM medium was -MEM without ribonucleosides and deoxyribonucleosides (Invitrogen) supplemented with BSA, pyruvate and glutamine, as described above. Cilostamide (8.5 µmol/l), dbcAMP (0.3 mmol/l), Hx (4 mmol/l), FSH (100 mIU/ml), EGF (10 ng/ml) and FF-MAS (10 µmol/l) were added alone or in combination, depending on the experimental protocol. Oocytes were matured for 20 h at 37°C in a 5% CO2 in air humidified atmosphere under the following conditions: (i) Hx; (ii) Hx + FSH, EGF or FF-MAS; (iii) dbcAMP; (iv) dbcAMP + FSH, EGF or FF-MAS; (v) cilostamide; (vi) cilostamide + FSH, EGF or FF-MAS.
For co-culture experiments, CEO were mechanically dissociated with a fine-bore Pasteur pipette after transfer to microwells, and NkO were matured together with their cumulus cells. Likewise in the cumulus-intact experiments, groups of 3040 dissociated cumulus complexes were cultured in 6080 µl of in vitro maturation medium (1 NkO/2 µl of medium) in 96-well plates.
At the end of maturation period, CEO were morphologically assessed for meiotic resumption, signified by GVBD, and extrusion of the PBI. GVBD and PBI rates were calculated on the basis of the original number of cultured GV stage oocytes.
Statistical analysis
Each experiment was repeated at least three times and frequencies of GVBD and PBI extrusion were expressed as mean±SEM. Data were compared by ANOVA and Duncan's test. P<0.05 was considered statistically significant.
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Results |
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Cilostamide treatment
In preliminary experiments, to establish optimal cilostamide concentration, a doseresponse curve (from 5 to 10 µmol/l) was performed, finding that an 8.5 µmol/l dose was able to maintain 89% of CEO at the GV stage. At such a concentration, GV arrest was completely reversible, as shown by the fact that, following 3 hr of preincubation with cilostamide, 95% of CEO underwent GVBD, and 57% of them extruded the PBI in medium not supplemented with the inhibitor. As illustrated in Figure 1C, small but non-significant increases in the GVBD rate were observed when oocytes were co-treated with FSH (17%), EGF (17%) or FF-MAS (19%). PBI emission was almost undetectable in all treated groups.
Meiotic maturation of NkO co-cultured with cumulus cells
In these experiments, induction of meiotic resumption was assessed in Hx- and dbcAMP-treated NkO co-cultured with their cumulus cells (Figure 2A and B respectively). Under these conditions, Hx inhibited GVBD to an extent comparable to that observed in intact CEO (32 and 25% respectively; P>0.05). Hx inhibition was overcome by culture in the presence of FSH (86%), EGF (71%), and FF-MAS (73%; P<0.01 versus Hx). EGF induced the extrusion of PBI in 50% of the oocytes, while treatment with FSH or FF-MAS did not raise PBI rates compared to the control group. Under repressive conditions imposed by dbcAMP, FSH and EGF stimulated GVBD to a similar extent (67 and 82% respectively. P<0.01 versus Hx). In both control and treatment groups, all oocytes that resumed meiosis were also able to extrude the PBI.
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Discussion |
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Whereas cumulus cells can generate both negative and positive influences on meiotic reinitiation, the nature of the prevailing positive signal triggered by FSH remains a matter of debate.
Recently, it has been proposed that FSH triggers in cumulus cells the sequential activation of PKC (Fan et al., 2003) and MAPK (Su et al., 2002
; Fan et al., 2003
) which, in turn, causes stimulation of the secretion of a GVBD-promoting soluble factor. The existence of such a paracrine factor is supported by experiments in which NkO co-cultured with their cumulus cells in the presence of Hx express high GVBD ability in response to FSH (Fan et al., 2003
). Indeed, our data are in agreement with these findings, as well as those reported by Byskov et al. (1997)
, although not entirely consistent with previous studies of Downs and collaborators, who considered unlikely the existence of a diffusible factor (Downs, 2001
). Those discrepancies can be attributed to different experimental conditions applied in the various experiments. In particular, the fact that we obtained an apparently higher percentage of NkO undergoing GVBD compared to the rate reported by Fan et al. (2003)
(86 and 50% respectively) may be explained with the application in our work of a lower oocyte-to-culture volume ratio, in the attempt to facilitate the concentration of released factors. In our hands, this system does not compromise oocyte viability, as demonstrated also by unaltered maturation rates compared to the culture in larger volumes of medium. Our experiments conducted in the presence of dbcAMP are also consistent with the involvement of a possible paracrine factor in the process of meiotic resumption, as shown by the fact that under such repressive conditions both FSH- and EGF-treated NkO co-cultured with cumulus cells also undergo GVBD and PBI extrusion. It should be noted however that, under cAMP suppression, physical dissociation of oocytes from cumulus cells appears to attenuate an intrinsic inhibitory influence on GVBD caused by the somatic compartment, as suggested by the lower maturation rate of cumulus-intact oocytes compared to co-cultured control groups (Figures 1B and 2B respectively). This has also been shown by Downs (2001)
who reported that, in the presence of Hx, dissociation of cumulus cells increases the rate of meiotic resumption in co-cultured oocytes. Further experiments presented by the same author appear to support the hypothesis that direct interaction between cumulus cells and the oocyte is functional to the maintenance of high levels of GVBD inhibition in the presence of Hx. In effect, addition of glycyrrhetinic acid, a gap junction inhibitor, increases in a dose-dependent fashion the ability of CEObut not NkOto undergo GVBD. In conclusion, our data are compatible with the existence of a stimulatory paracrine factor derived by the stimulation of cumulus cells, considering the highly significant difference (P<0.01) in the GVBD rate of the co-cultured control group compared to the FSH or EGF treatments. However, this question remains controversial, since other studies (Downs, 2001
) have been unable to demonstrate the capacity of FSH to improve the GVBD frequency in NkO co-cultured with their cumulus cells after stimulation with FSH. Finally, it should not be underestimated that culture conditions, and in particular culture volumes, can influence the effect of cumulus cells on the control of meiotic resumption. This is discussed by Downs (2001)
, with reference to the fact that in his system40 µl drops under oilthe meiosis-inducing effect of cumulus cell co-cultures was lower compared to the one supported by the systemwells containing 400 µl of medium without oil overlayused by Byskov et al. (1997)
.
In relation to PBI emission, from our results it is also evident that in CEO cultured in the presence of Hx, FF-MAS supports PBI emission to a lower extent than FSH or EGF. It may be argued that, after 20 h of culture, a low PBI rate following FF-MAS treatment is caused by slower kinetics of GVBD, whose rate increases only after 10 h of incubation (Hegele-Hartung et al., 1999). But in reality, under conditions identical to those applied for testing FF-MAS, FSH-stimulated meiotic resumption does not increase significantly before 12 h (Su et al., 2002
). Therefore, rather than being the consequence of slow kinetics of meiotic resumption, the restricted ability to sustain the completion of the meiotic process would appear as an intrinsic attribute of FF-MAS. Recent experiments (Cukurcam et al., 2003
) have further clarified FF-MAS action. In particular, it appears that, in mouse in vitro matured oocytes, this agent reduces the incidence of precocious chromatid separation caused by suboptimal culture conditions and the absence of cumulus cells. However, when meiotic maturation occurs in the presence of Hx, such a protective effect is accompanied by a high frequency of diploid MII chromosome complements. This suggests that in vitro, while stimulating normal nuclear maturation to MII under repressive conditions, FF-MAS may not be sufficient to induce the concomitant process of cytokinesis. Consistent with these findings, our experiments indicate that, under continued inhibitory conditions, the GVBD to PBI transition is not a default process, but requires specific regulatory cues originating from cumulus cells. The mere presence of these cells does not represent a stimulus sufficient for the extrusion of the polar body, considering that in our experiments the FF-MAS treatments were also conducted using CEO. So, stimulation of the somatic compartment is critical. Furthermore, the fact that, after FSH treatment in co-cultured NkO, arrested at the GV stage with Hx, the rate of PBI extrusion is lower compared to that of the CEO groups matured under otherwise identical stimulation conditions indicates that under the influence of this inhibitor direct oocyte somatic cell contacts are also important for meiotic progression beyond GVBD. Also, with reference to these experiments it does not appear obvious why EGF expresses a higher PBI-inducing activity compared to FSH. However, this may reflect the fact that the signalling pathways of these two meiosis-promoting agents are not identical, as shown by the fact that via the stimulation of cumulus cells FSH, but not EGF, causes an increase in the oocyte cAMP concentration (Webb et al., 2002
). Contrary to the evidence derived from the treatment with Hx, in the presence of dbcAMP, emission of PBI occurs with a high frequency in FSH-treated co-cultured NkO, once again emphasizing the point that in vitro meiotic outcome depends not only on the nature of the activating stimulus but also on the conditions used to ensure meiotic arrest.
In conclusion, in this paper we demonstrate that the use of diverse meiotic inhibitors can discriminate between the relative activity of different meiotic inducers. In mouse CEO maintained in meiotic arrest with Hx, FF-MAS expresses a GVBD-inducing ability equivalent to FSH and EGF. By contrast, experiments conducted in the presence of dbcAMP suggest that FSH and EGF can stimulate cumulus cells to produce a meiosis-promoting signal that does not necessarily coincide with FF-MAS. In addition, inability of FSH, EGF, and FF-MAS to induce GVBD in the presence of the PDE 3 inhibitor cilostamide confirms that, irrespective of the action of a positive stimulus, regulation of intra-oocyte cAMP degradation is essential for meiotic resumption. Finally, our results demonstrate that stimulation of the somatic cell compartment and maintenance of its association with the oocyte appears also to control positively the emission of PBI stimulated by FSH.
Although the oocyte plays a central role in the regulation of meiotic maturation (Su et al., 2002) the identification of the signal(s) by which gonadotrophin-stimulated cumulus cells instruct the oocyte to restart and complete the meiotic process could provide important clues to improve the competence of in vitro matured mammalian oocytes.
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Acknowledgements |
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References |
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Submitted on February 4, 2004; resubmitted on July 8, 2004; accepted on August 18, 2004.