Hum. Reprod. Advance Access originally published online on March 20, 2006
Human Reproduction 2006 21(7):1765-1770; doi:10.1093/humrep/del074
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Meiotic spindle configuration is differentially influenced by FSH and epidermal growth factor during in vitro maturation of mouse oocytes
1 Department of Biomedical Sciences and Technologies 2 Department of Experimental Medicine, University of L’Aquila, L’Aquila 3 Department of Histology and Medical Embryology, University of Roma, ‘La Sapienza’, Roma, Italy
4 To whom correspondence should be addressed at: Department of Biomedical Sciences and Technologies, University of L’Aquila, Via Vetoio, Loc. Coppito, 67010 Coppito, L’Aquila, Italy. E-mail: cecconi{at}univaq.it
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussion Acknowledgements References |
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BACKGROUND: To ascertain whether different hormonal treatment protocols could affect metaphase II (MII) spindle morphology, meiotic spindle organization was detected in prepubertal mouse oocytes matured under conditions allowing spontaneous, FSH- or epidermal growth factor (EGF)-dependent meiotic maturation. METHODS: Oocyte-cumulus complexes (OCCs) were matured either spontaneously (control; n = 270) or in the presence of hypoxanthine (Hx) plus FSH (n = 400) or EGF (n = 370). Spindles were detected by immunofluorescence analysis. In vivo ovulated (IVO) oocytes were processed similarly. RESULTS: IVO oocytes displayed spindles underlying the oolemma and with focused poles marked by spots of
-tubulin, whereas the majority (89%) of control oocytes had barrel-shaped spindles, positioned away from the oolemma, and with -tubulin distributed along microtubules. Similar configuration/localization was found in 85% of the oocytes matured in vitro in the presence of Hx and FSH. In the presence of Hx-EGF, 35% of the oocytes showed spindles with an IVO-like configuration, although -tubulin was homogeneously distributed throughout microtubules. Independently of spindle shape, 52% of EGF-stimulated oocytes had spindles positioned near the oolemma, in comparison to just 24% of FSH-treated and 13% of control oocytes. CONCLUSIONS: These results indicate that FSH and EGF can differently affect meiotic spindle morphology, and that EGF might be a stronger contributor than FSH to the acquisition of oocyte competence.
Key words: EGF/FSH/ in vitro maturation/oocyte meiotic spindle
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussion Acknowledgements References |
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With respect to the time-length of the growth phase, in all mammalian oocytes meiotic maturation lasts a short interval, during which a very complex series of molecular, cytoplasmic and nuclear events must take place in a co-ordinated way, in order to assure the production of fertilizable metaphase II (MII)-arrested oocytes.
In vitro, meiotic resumption, evidenced by germinal vesicle breakdown (GVBD), can be induced either as a spontaneous or as a gonadotropin-dependent process. The former occurs as a consequence of oocyte removal from follicular inhibitory environment (Pincus and Enzmann, 1935
), while the latter is induced by gonadotropic action on cumulus cells, that produce meiosis-inducing signal(s) capable of overriding meiotic arrest (Downs et al., 1988; Su et al., 2002; Fan and Sun, 2004). In both processes, a key step is represented by decrease in intracellular cAMP (Dekel et al., 1988; Horner et al., 2003) and by subsequent activation of M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAPK) pathway (Fan and Sun, 2004). FSH and LH, alone or in combination (Thomas et al., 2003), as well as epidermal growth factor (EGF) (DeLafuente et al., 1999; Coticchio et al., 2004) are utilized to induce complete meiotic maturation up to emission of the first polar body (PB1) in cultured oocyte-cumulus complexes (OCCs). Cumulus cells mediate hormonal response, because of the presence of specific receptors (Ulloa-Aguirre et al., 1995), and contribute also to acquisition of oocyte capability to reach MII stage, to be fertilized and to undergo embryonic development (Vanderhyden and Armstrong, 1989; Cecconi et al., 1996; Thomas et al., 2003; Combelles et al., 2004, 2005). Indeed, a bidirectional communication exists through which cumulus cells and oocytes regulate reciprocal functions by a paracrine and gap junction mediated signalling (Albertini et al., 2001; Eppig, 2001). Such an axis results in the activation of metabolic and regulative pathways involved in the acquisition of oocyte developmental competence (Su et al., 2002, 2004; Coticchio et al., 2004; Combelles et al., 2005).
In vitro maturation (IVM) is efficiently used to obtain MII-arrested mouse oocytes that are competent to be fertilized and capable of producing viable embryos (Albertini, 2003; Wassarman et al., 2005). Although in some large mammals, like the cow, IVM is used to increase the number of embryos to be transferred (Barnes et al., 1993; Bousquet et al., 1999), in humans this procedure should be considered still experimental despite some promising preliminary results (Mikkelsen et al., 1999; Combelles et al., 2005). Cumulus cell removal, which is necessary to allow scoring of oocyte nuclear status at retrieval, could contribute to the accumulation in these oocytes of M-phase deficiencies at the end of the culture period (Combelles et al., 2002). In fact, a recent paper by Combelles et al. (2005) demonstrated that human oocytes co-cultured with their dissociated cumulus cells in a 3D-gel system exhibited increased MAPK activity and maintained MII arrest more efficiently than denuded oocytes, despite comparable rates of maturation to MII. Thus, among the parameters utilized to assess oocyte developmental capacity, the ability to resume and complete meiotic maturation is not sufficient per se. The possibility to use spindle analysis as a parameter to assess oocyte quality derives from studies conducted on oocytes of various mammalian species (Hu et al., 2001; Eichenlaub-Ritter, 2002; Albertini, 2003; Plancha, 2005) and has been recently utilized also to assess effects of toxicants and drugs (Eichenlaub-Ritter and Boll, 1989; Can and Semiz, 2000; Rossi et al., 2006). In mice, in vivo ovulated (IVO) and IVM oocytes show significant differences in meiotic spindle shape and size (Hu et al., 2001; Sanfins et al., 2003). These differences have been related to animal age (Hu et al., 2001) as well as to asynchrony in the process of nuclear and cytoplasmic maturation (Eppig and O’Brien, 1998; Mermillod et al., 1999). This could be attributed to the lack of suitable culture conditions that can dramatically impair oocyte quality (Eichenlaub-Ritter and Peschke, 2002; Hussein et al., 2005). Indeed, in humans Cekleniak et al. (2001) have demonstrated that a modification in standard culture medium composition significantly improves PB emission rate and the number of oocytes with a normal spindle organization.
In this work we confirmed and extended previous observations by Sanfins et al. (2003) by comparing meiotic spindle organization in cumulus enclosed oocytes that were matured under conditions allowing spontaneous, FSH- or EGF-dependent meiotic maturation. The aim was to ascertain whether different hormonal supplementations could affect the morphology of the MII spindle.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussion Acknowledgements References |
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Animals
All experiments were performed using 23- to 25-day-old female immature Swiss CD1 mice (Harlan Italy, Udine, Italy) maintained on a 12:12 h light : dark regimen and at room temperature of 21 ± 1°C. Animal care and handling were approved by the Animal Care Committee of the University of L’Aquila, in accordance with the procedures described in the ‘Guidelines for the care and use of laboratory animals’ (NIH Guide).
Chemicals
All chemicals were of the purest analytical grade and were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise indicated.
Collection of oocytes matured in vivo and in vitro
To obtain IVO oocytes, animals were primed with 5 IU of pregnant mare’s serum gonadotrophin (PMSG; Intervet Italia, Milan, Italy) and with 5 IU of hCG (Serono International S.A., Geneva, CH) 48 h later. About 14–15 h post-hCG, MII-arrested oocytes, released from the oviductal ampullae, were collected into Hepes-buffered Eagle’s minimal essential medium (MEM) (Invitrogen, Paisley, UK), supplemented with 0.23 mM pyruvic acid, 2 mM L-glutamine, 0.3% bovine serum albumin (BSA) (collection medium). Cumulus cells were removed by brief treatment (1 min) with hyaluronidase (1 mg/ml) at room temperature.
For IVM experiments, OCCs were collected from the ovaries of primed mice and incubated in MEM alpha medium (Invitrogen) supplemented with 0.3% BSA, 0.23 mM pyruvic acid, 2 mM L-glutamine. This medium was used for spontaneous maturation experiments (control). To assess hormone-induced maturation, medium was supplemented with FSH (100 mU/ml) or EGF (10 ng/ml), and with 4 mM Hypoxanthine (Hx; Coticchio et al., 2004). IVM was performed in a humidified atmosphere of 5% CO2 at 37°C, for 17–18 h (control) or for 20 h when culture medium was supplemented with Hx. At the end of the culture period, cumulus cells were removed by gentle pipetting. In some experiments, OCCs were briefly incubated in hyaluronidase before removing cumulus cells similarly to IVO oocytes.
Only oocytes arrested at MII, recognized by extrusion of PB1, were further analysed.
Immunofluorescence analysis
IVO (n = 180) and IVM oocytes matured under the different experimental conditions (control, spontaneously matured n = 270; Hx + FSH n = 400; Hx + EGF n = 370) were labelled according to the protocol described by Sanfins et al. (2003). Oocytes were fixed for 30 min at 37°C in a microtubule-stabilizing buffer, containing 2% formaldehyde, 0.1% Triton X-100, 1 µM taxol, 10 IU/ml aprotinin and 50% deuterium oxide (Cekleniak et al., 2001). Oocytes were stored at 4°C until further processing in a blocking solution of phosphate-buffered saline (PBS) containing 1% BSA, 0.2% powdered milk, 2% normal goat serum, 0.1 M glycine, 0.01% Triton X-100 and 0.2% sodium azide (blocking solution). Then, germ cells were incubated in the presence of a mouse monoclonal anti-
,
-tubulin (1:150; Sigma) followed by Texas Red dye conjugated AffinyPure donkey anti-mouse IgG (1:100; Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA) for 1 h at 37°C each. To detect -tubulin distribution over the spindle, oocytes were labelled with rabbit monoclonal anti--tubulin (1:400; Sigma), followed by anti-rabbit IgG-FITC (1:50) for 1 h at 37°C each. Finally, the oocytes were labelled with Hoechst 33342 (1 µg/ml) in blocking solution for 10 min at room temperature and mounted on slides. Oocytes were analysed using a fluorescence microscope (AxioPlan 2, Zeiss; x40 objective) with digital images collected with Leica DFC350 FX camera interfaced with IM500 Leica software. Spindles were classified into ‘focused’, when spindle had pointed poles, and ‘barrel’, when spindle had large poles. The number of cytoplasmic microtubule-organizing centres (MTOCs) was evaluated by focusing through the entire oocyte volume (Sanfins et al., 2003).
Statistical analysis
All the experiments were repeated at least three times. Data were expressed as the mean ± SEM and compared by ANOVA followed by Bonferroni test, or by paired Student’s t-test for comparison of data derived from two groups (InStat 2.03, GraphPad Software for a Science, CA, USA). Percentages were compared by chi-square analysis. Values with P < 0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussion Acknowledgements References |
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Meiotic maturation
OCCs were matured in vitro spontaneously (control), or in the presence of 100 mU/ml FSH or 10 ng/ml EGF, with or without 4 mM Hx. Under all experimental conditions, oocytes underwent GVBD and extruded the PB1 with an efficiency comparable to IVO oocytes, and without any difference among treatments (Figure 1) (Coticchio et al., 2004
).
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Spindle analysis
Spindle organization was analysed in IVO and IVM oocytes cultured as reported above. As shown in Figure 2, a high percentage (mean ± SEM; 96 ± 1%) of IVO oocytes showed bipolar spindles with tightly focused poles (Figure 3A) and spots of
-tubulin (Figure 3B).
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In spontaneous IVM oocytes, 89 ± 2% of the spindles were barrel-shaped (Figure 3C), and only 8 ± 1% displayed bipolar spindles with focused poles;
-tubulin appeared homogeneously distributed throughout the spindle (Figure 3D).
When IVM was carried out in the presence of Hx and FSH, the majority of cultured oocytes (85 ± 2%) had spindles characterized by a large barrel configuration (Figure 4A). By contrast, in the presence of Hx and EGF, the percentage of oocytes with a convergent array of microtubules at the spindle poles increased significantly up to 35 ± 2% (Figure 4B) (P < 0.05), and those with barrel aspect decreased to 63 ± 2%. Analysis of -tubulin distribution revealed that all the FSH (Figure 4C) and EGF-stimulated oocytes (Figure 4D) showed this protein distributed throughout the spindle. Independently of in vivo or in vitro maturation, all the oocytes had chromosomes well aligned at the equator.
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About 3–4% of the oocytes, under all in vitro experimental conditions, showed spindles with a completely abnormal structure characterized by dispersed microtubules and chromosomes (data not shown).
In addition we found that, at MII, 100% of IVO oocytes showed the spindle localized near the oolemma, whereas this positioning was attained by only 13% of spontaneously matured, 24% of FSH- and 52% of EGF-stimulated oocytes (n = 100/group; P < 0.001). Hyaluronidase treatment did not affect these morphological aspects (data not shown).
The number of cytoplasmic MTOCs recorded was strictly related to spindle array and not to the culture conditions. A mean number of 11 ± 4 MTOCs was detected in the presence of barrel spindles, and 27 ± 5 MTOCs in the presence of pointed bipolar spindles with focused poles (P < 0.05; n = 30 oocytes/experimental group).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussion Acknowledgements References |
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This study demonstrates that the selection of specific IVM conditions can affect meiotic spindle configuration in prepubertal mouse oocytes differently.
This analysis after in vivo and in vitro oocyte maturation has been performed by several groups. Hu et al. (2001) suggested that spontaneously matured denuded oocytes from prepubertal animals had rather more pointed poles in comparison to those matured in vivo. Contrasting results have been obtained by Sanfins et al. (2003). These authors assessed spindle behaviour in naturally ovulated oocytes, IVO oocytes obtained from standard superovulation protocols and cumulus cell-oocyte complexes undergoing spontaneous meiotic maturation. They found that, while the IVO oocytes had normal bipolar spindles at MII stage, with focused poles characterized by the presence of distinct -tubulin foci, the IVM group exhibited barrel-shaped spindles with fewer acetylated microtubules and -tubulin diffusely distributed along the microtubules. Moreover, in contrast to IVO oocytes, IVM oocytes showed perturbation in the process of anchoring the spindle in close proximity to the cortex with the spindles positioned away from the oolemma (Deng et al., 2005; Plancha, 2005). Differences in experimental protocols can account for these discrepancies. Our results on IVO and spontaneously matured oocytes are in agreement with those by Sanfins et al. (2003). Similar to their work, we utilized intact complexes and the same fixation protocol. Besides differences between studies, it appears evident that spontaneous maturation does not assure coordination between nuclear and cytoplasmic maturation. Therefore, it is important to obtain improvement in the in vitro conditions.
IVM in the presence of Hx and FSH is commonly used to induce a better synchrony in the process of nuclear and cytoplasmic maturation. In our experiments, beside the oocytes undergoing spontaneous maturation, also those undergoing FSH-induced maturation showed a very high percentage (85%) of spindles with the barrel-shaped appearance. The concentration of FSH that was chosen (100 mU/ml) is commonly used for these kinds of experiments (Su et al., 2002; Coticchio et al., 2004). Yet, when FSH was used at lower or higher concentrations we did not observe improved spindle morphology (Roberts et al., 2005; unpublished results). Thus, our results demonstrate that although FSH mimics an in vivo process, promoting cumulus expansion and oocyte maturation in vitro (Salustri et al., 1990), it is unable to induce all the changes that occur in vivo.
It has been proposed that the barrel-shaped aspect results from the incorporation in the spindle of an excessive number of MTOCs, which significantly reduces the stores of cytoplasmatic -tubulin in the mature oocyte (Combelles and Albertini, 2001; Sanfins et al., 2003, 2004; Plancha, 2005). Such an abnormal redistribution could significantly reduce the stores of available maternal -tubulin, necessary to sustain normal embryonic development (Yuba-Kubo et al., 2005). In FSH-stimulated oocytes, we observed high recruitment in the spindle of MTOCs from the cytoplasm, as evidenced by the decreased number of cytoplasmic MTOCs. The number of cytoplasmic MTOCs was comparable to that observed in spontaneously matured oocytes and lower than that recorded in IVO oocytes. Moreover, in FSH-stimulated oocytes also -tubulin distribution was similar to that of spontaneously matured oocytes (this study; Sanfins et al., 2003, 2004), and this protein appeared distributed throughout the spindle without clearly recognizable polar spots typical of IVO oocytes. Moreover, the finding that only 24% of these oocytes displayed normal positioning of spindles near the oolemma, compared with 100% of IVO oocytes, supports the hypothesis that, during IVM, FSH is not involved in the acquisition of normal oocyte polarity.
Concerning EGF, it is well known that during the periovulatory period, EGF and EGF-like growth factors act as paracrine mediators of LH/hCG stimulation of follicles (Park et al., 2004). In vitro, EGF influences cumulus expansion, meiotic maturation and oocyte developmental competence (Downs et al., 1988; DeLafuente et al., 1999; Ashkenazi et al., 2005; Conti et al., 2005), by a signalling cascade specifically mediated by cumulus cells (Jamnongjit et al., 2005). Here we show that 36% of the oocytes matured in the presence of EGF display spindles with focused poles and with a number of cytoplasmic MTOCs similar to that detected in IVO oocytes. This result suggests that EGF may activate process(es) regulating MTOCs recruitment from the cytoplasm and also microtubule tethering into poles. The fact that only a part of EGF-stimulated OCCs have spindles with the IVO-like array suggests that, at retrieval, the population of preovulatory oocytes is heterogeneous, and only in part capable of proper response to stimuli. This possibility is supported also by results by O’Donnell et al. (2004), who analysed EGF-induced calcium release from intracellular stores of cumulus cells from mouse OCCs. They found that only a part of OCCs responded to EGF stimulation, and proposed that in vivo this mechanism may be important to activate only a competent and selected population of OCCs.
The above-mentioned heterogeneity could determine, at the end of culture, a mixture of ‘unaged’ and ‘aged’ MII oocytes, which can be evidenced by spindle detachment from oolemma during meiotic arrest. By analysing spindle positioning we found that, independently of final array, 52% of the oocytes matured in the presence of EGF had spindles localized near the oolemma, as described for all the IVO oocytes. The fact that this ‘normal’ positioning was more represented in the EGF than in FSH group (24%) suggests that the beneficial effects of EGF might be related to a more synchronous rate of maturation, resulting in a higher percentage of IVO-like spindles in comparison with FSH-stimulated oocytes.
This result reinforces the hypothesis that EGF, by still unknown modalities, may contribute to the acquisition of normal oocyte polarity. However, despite the presence of focused spindles, in all the EGF-stimulated oocytes -tubulin has been detected along microtubules, in a pattern similar to that described for FSH. Thus, even if EGF can induce proper focusing of poles and spindle positioning in a selected population of oocytes, it is unable to regulate the normal distribution of -tubulin. All these observations confirm that IVM per se is a process in which altered regulation of some important steps of cell cycle is likely (Plancha, 2005). Although in our experiments oocytes matured with either FSH or EGF have comparable maturation rate up to MII, DeLafuente et al. (1999) reported that the presence of EGF during IVM increased significantly not only the percentage of oocytes reaching MII, but also blastomere proliferation. This report, together with our results, suggests that EGF could regulate better than FSH the induction of some of the molecular processes associated with cytoplasmic maturation and embryonic development, at least during IVM.
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Acknowledgements |
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We thank Dr Mauro Maccarrone for critical reading of the manuscript. The work was supported by funds provided by MIUR (PRIN 2003) to S.C. and by the University of Rome ‘La Sapienza’ 2004 to R.C.
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Submitted on December 23, 2005; resubmitted on February 13, 2006; accepted on February 17, 2006.
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