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Endocrinology, doi:10.1210/en.2007-0874
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Endocrinology Vol. 149, No. 1 100-107
Copyright © 2008 by The Endocrine Society

Meiotic Maturation of Incompetent Prepubertal Sheep Oocytes Is Induced by Paracrine Factor(s) Released by Gonadotropin-Stimulated Oocyte-Cumulus Cell Complexes and Involves Mitogen-Activated Protein Kinase Activation

Sandra Cecconi, Annunziata Mauro, Giulia Capacchietti, Paolo Berardinelli, Nicola Bernabò, Anna Rita Di Vincenzo, Mauro Mattioli and Barbara Barboni

Department of Biomedical Sciences and Technologies (S.C.), University of L’Aquila, 67100 L’Aquila, Italy; and Department of Comparative Biomedical Sciences (A.M., G.C., P.B., N.B., A.R.D.V., M.M., B.B.), University of Teramo, 64100 Teramo, Italy

Address all correspondence to: Prof. Sandra Cecconi, Department of Biomedical Sciences and Technologies, University of L’Aquila, 67100 L’Aquila, Italy. E-mail: sandra.cecconi{at}cc.univaq.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, sheep oocyte-cumulus cell complexes (OCC) derived from medium (M) antral follicles (M-OCC) were in vitro matured alone or in coculture with OCC derived from small (S) antral follicles (S-OCC) to investigate the contribution of cumulus cells (CC) and oocytes to the process of oocyte meiotic maturation and cumulus expansion (CE). Experiments were conducted with or without gonadotropins (FSH/LH). Regardless of culture conditions, about 12% of S-oocytes reached the metaphase II stage, and S-CC showed a low degree of CE. In contrast, both maturational processes were significantly stimulated by gonadotropins in M-OCC. However, about 48% of S-oocytes progressed to metaphase II, and S-CC expanded after coculture with gonadotropin-stimulated M-OCC and M-CC but not with mural granulosa cells. Both maturational processes were inhibited when S-OCC were cocultured with M-denuded oocytes, or when S-denuded oocytes were cocultured with M-CC. The capacity of these paracrine factor(s) to activate the MAPK pathway in somatic and germ cells of S-complexes was investigated. It was found that MAPK kinase/MAPK phosphorylation levels in M-OCC but not in S-OCC were significantly increased by gonadotropins, first in CC and later in the oocytes. Kinase phosphorylations were activated only in S-oocytes cocultured with M-OCC or M-CC. These results demonstrate that soluble factors specifically produced by M-CC are capable to induce meiotic maturation and CE in S-complexes by acting via CC. These factors can induce MAPK activation only in S-oocytes, whose meiotic arrest could be due to the inability of surrounding CC to respond to gonadotropin stimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE KEY EVENT in female reproductive physiology is oogenesis, a long growth and maturation process by which the oocyte acquires sequentially the capacity to direct meiotic maturation and to support fertilization and embryo development.

The fact that oocyte growth and development occurs within the follicle highlights the crucial role exerted by somatic compartment in sustaining this process. Indeed, besides exerting a nutritional role (1), granulosa cells mediate gonadotropin stimulation and the transfer of messenger molecules to the oocyte (2, 3). However, because the oocyte controls many granulosa cell functions, a regulatory loop exists by which granulosa cells and oocyte control reciprocal functions either through gap junctions or by paracrine factors (4, 5, 6, 7).

In this context, the oocyte acquires meiotic competence as a result of a number of timely orchestrated events involving nuclear remodeling and specific changes occurring within the cytoplasm (8, 9, 10, 11). The acquisition of full meiotic competence occurs in a step-wise manner during oogenesis, as demonstrated by the finding that the oocyte first becomes capable of undergoing germinal vesicle breakdown (GVBD) and then of completing meiosis up to metaphase II (MII) (4, 12). Meiotic maturation can be induced by removing the oocyte from the inhibitory follicle microenvironment (spontaneous maturation) (13) or by triggering the process with LH, as occurs under physiological conditions (14), or with FSH/LH, as occurs for intact follicle culture (15). Under hormonal stimulation, a specialized population of granulosa cells surrounding the oocyte, the cumulus cells (CC), mediates the production and/or transfer of meiosis-inductive signal(s) to a germ cell ready to undergo cell cycle resumption (12, 16, 17, 18, 19).

Even if all the molecular mechanisms involved in the process of hormone-dependent maturation are still incompletely understood (20, 21, 22, 23), evidence shows that, in many mammalian species, gonadotropins stimulate CC to synthesize molecules able to drive GVBD as meiosis-activating sterols (24). In mice, Su and collaborators (22) demonstrated that the preovulatory oocyte plays a key role in promoting resumption of meiosis. Indeed, the germ cell secretes soluble factor(s) activating in gonadotropin-stimulated CC the production of a meiosis-inducing signal that is transferred back to the oocyte via gap junctions. Resumption of meiosis requires the activation of MAPK in CC but not in the oocyte (18, 22, 23, 25, 26). In fact, the inhibition of MAPK activation in stimulated CC blocks the induction of GVBD and cumulus expansion (CE) in both mos+/+ and mos–/– oocyte-cumulus complexes (OCC) (22). Also, in porcine oocytes, the activation of MAPK in CC and not in the oocyte is essential for GVBD (20, 27).

Gonadotropins are necessary also to promote the process of CE through the activation of specific genes in the somatic compartment of the preovulatory complex (28, 29). In the mouse, CE is stimulated by factors specifically secreted by the fully grown oocyte (CE-enabling factors) (30) via activation of MAPK3/1 and MAPK14 (23, 29, 31, 32). By contrast, in the pig, this process is considered to be oocyte-independent and strictly related to the autocrine secretion of CE-enabling factors by CC (30, 33).

Due to the complexity of the molecular mechanisms underlying gonadotropin-induced maturation, OCC collected from follicles at different phases of antral development could represent a useful tool to evaluate the progressive activation of the molecular pathways controlling meiotic maturation and CE. Starting from these considerations, we used two classes of prepubertal sheep OCC derived from small (S) (follicle diameter < 1 mm) and medium (M) (follicle diameter 3–4 mm) antral follicles. In contrast with M-OCC, oocytes from S-follicles were unable to mature after gonadotropin stimulation (15, 34). In the present study, S- and M-complexes were cultured together to evaluate whether somatic and germinal compartments of M-complexes could be able to release factor(s) stimulating meiotic maturation CE as well as MAPK activation in S-OCC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
All the chemicals used in the present study were purchased from Sigma Chemical Co. (St. Louis, MO), unless otherwise indicated.

Animals
Ovaries collected from prepubertal sheep (3–6 months of age), grown under controlled nutritional regimens (35), and slaughtered during the breeding season, were maintained during transport to the laboratory (15–30 min) at room temperature. Ovaries were dissected from the rest of the reproductive tract and rinsed several times in PBS supplemented with antibiotics (75 mg/liter penicillin-G, 50 mg/liter streptomycin sulfate).

Collection and classification of OCC
Antral follicles (S, 0.5–1 mm; M, 3–4 mm) were mechanically isolated from thin strips (about 15 mm long, 2 mm wide) of cortical ovarian tissue and classified as healthy on the basis of their morphology (translucency, lack of free particles within antral cavity, and the presence of blood vessels). From each follicle-sized group, released OCC with compact cumuli and homogeneous cytoplasm were chosen for further culture and designated as S-OCC or M-OCC, respectively. Then oocyte diameters (zona pellucida excluded) were recorded for either S-OCC or M-OCC using a calibrated eyepiece of an inverted microscope. The mean oocyte diameter recorded for the oocytes of the S-OCC (S-oocytes) was 110 ± 3 µm and for those from M-OCC (M-oocytes) was 120 ± 3 µm. When needed, OCC were mechanically deprived of surrounding CC, and the denuded oocytes (DO) or CC obtained from each size group were used depending on experimental protocol. M- and S-complexes were in vitro matured either alone or together following the experimental design reported in Fig. 1Go.


Figure 1
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  		FIG. 1. Schematic representation of experimental culture conditions. A and a, S-OCC or DO (n = 55) and M-OCC or DO (n = 40); B, S-OCC (n = 15) Co-IVM with M-OCC (n = 30); C and D, S-OCC (n = 15) Co-IVM with M-DO (n = 30) or M-CC; b–d, S-DO (n = 15) Co-IVM with M-OCC (n = 30), M-DO (n = 30), or M-CC, respectively. See text for details.

 
In vitro maturation conditions
In all experiments performed, a fixed ratio between oocyte volume and culture medium was maintained. The volume of culture medium was identified on the basis of preliminary experiments aimed to establish the minimum volume not affecting CE and meiotic maturation rate. In a first series of experiments, M-OCC (n = 40) or S-OCC (n = 55) were cultured in 60 µl TCM 199 supplemented with 10% fetal calf serum with or without gonadotropins (0.1 UI/ml ovine FSH and/or 0.1 UI/ml porcine LH, USDA-pLH-B-1; National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD; see Fig. 1Go, A and a) in 96-well flat-bottom plates (NUNC). In vitro maturation (IVM) was carried out for 24 h at 38.5 C in 5% CO2 in air. At the end of IVM, CE was assessed before denuding oocytes of surrounding cumulus-corona cells (3–4 min in 0.1% hyaluronidase). Oocyte nuclear stage was analyzed as described in the following. The same experimental procedure was used for M- and S-DO (Fig. 1Go, A and a).

In a second series of experiments, S-OCC (n = 15) were cocultured with M-OCC (n = 30; see Fig. 1BGo) in 60 µl TCM 199 supplemented with 10% fetal calf serum and with FSH and/or LH for various times, ranging from 0–24 h, under the same experimental conditions described above. These experimental conditions are indicated as Co-IVM. On the basis of preliminary experiments, the ratio S-OCC/M-OCC was selected among other conditions tested (30 S-OCC plus 15 M-OCC, 23 S-OCC plus 25 M-OCC).

To elucidate the role played by the germinal and somatic compartments during the process of meiotic maturation, Co-IVM experiments were carried out by incubating M-DO or M-CC with S-OCC (Fig. 1Go, C and D) or with S-DO (Fig. 1Go, b–d). To evaluate specificity of CC, S-OCC were cocultured with mural granulosa cells obtained from medium antral follicles following the protocol established by Mattioli (19). After determining cell viability by Trypan blue dye exclusion method, somatic cells were plated at the final concentration of 0.8 x 106 cells/ml, a concentration similar to that of CC. Finally, 15 S-OCC were added and cultured as described above. At the end of coculture experiments, oocyte diameters were measured again to monitor the lack of cross-contamination between the two experimental groups.

Evaluation of CE
At the end of culture period, CE was recorded in M-OCC and S-OCC cultured in the different experimental conditions (IVM and Co-IVM). To verify whether S-oocytes could stimulate the process of CE, S-OCC were microsurgically deprived of the oocyte by using a micromanipulation apparatus as described by Buccione et al. (36). The resulting oocytectomized complexes (OOX) are indicated as S-OOX. According to the experimental design, 15 S-OOX were cocultured with 30 M-OCC or M-CC, and CE was monitored 24 h later.

In our study, CE evaluation was performed according to Vanderhyden et al. (30), but data were arbitrarily grouped into three main classes: low degree (no detectable expansion and CC spreading/presence of very few CC attached to the substratum), moderate degree (expansion in the outer layers of CC), and high degree (all the layers except the innermost corona radiata expanded/all the CC surrounding the oocyte expanded, as observed after in vivo maturation).

Assessment of oocyte meiotic maturation
After 24 h of culture, oocyte nuclear stage was determined by staining germ cells with Hoechst 33342 (10 µM in culture medium for 20 min) (37) and by observing them under an inverted microscope (Eclipse 600, Nikon). To obtain oocytes at defined stages of meiosis, germ cells were removed from culture medium in coincidence with the major events of meiotic progression: germinal vesicle (GV) at 0 h, GVBD at 8 h, MI at 16 h, and MII at 24 h. The stage of meiotic progression was assessed as described above. Oocytes with the same nuclear stage and their dissociated CC were placed in 5 µl ice-cold collection buffer (2% SDS supplemented with 2 mM sodium orthovanadate and 10 mM sodium fluoride) and stored at –70 C until analysis.

Western blot analysis
Phosphorylated (p)/active forms of MAPK kinase (MEK) and MAPK (pMEK and pMAPK) were detected by Western blot analysis in the somatic and germinal compartments of M- and S-OCC. The analysis was performed on oocytes collected at definite meiotic stages (i.e. 0, 8, 16, and 24 h of culture) and on related CC. For protein extraction, 30 µl of a 2% SDS solution containing 2 mM phenylmethylsulfonyl fluoride, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 10 µg/ml leupeptin, 10 µg/ml antipain, and 100 U/ml aprotinin was added to each sample. Protein content was evaluated by the Lowry method (38). Proteins (30 µg/sample) were then separated by 10% SDS-PAGE and then electrophoretically transferred onto nitrocellulose (Hybond C Extra; Amersham Pharmacia Biotech, Inc., Piscataway, NJ) according to standard procedures. The following primary antibodies were used: monoclonal anti-pERK1/2 (1:4000; Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal anti-ERK2 (1:4000; Santa Cruz), polyclonal anti-pMEK1/2 (1:1000; Cell Signaling Technology, Inc., Danvers, MA), and monoclonal anti-{alpha}-tubulin clone B-5-1-2 (1:3000). Membranes were then incubated with peroxidase-conjugated antimouse IgG or antirabbit IgG (1:3000; Santa Cruz) and finally processed using ECL Western blot analysis system (Amersham Bioscience GmbH, Freiburg, Germany).

Statistical analysis
All experiments were carried out least three times in independent trials (five replicates per group per experiment). About 250 oocytes were analyzed for each time point. For CE and meiotic stage experiments, about 80–100 complexes were analyzed for each experimental condition. All data are expressed as mean percentage ± SEM. The results of meiotic maturation were compared by ANOVA followed by Tukey’s test (version 2.0; Orion Software Development, Longmont, CO). Percentages were compared by {chi}2 analysis. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CE and meiotic maturation of S- and M-OCC
OCC derived from medium (M-OCC) and small (S-OCC) antral follicles displayed a different capacity to undergo CE (Fig. 2Go, A and B) and oocyte maturation (Fig. 3AGo). Assessment of CE revealed that in the presence of FSH/LH or FSH alone, more than 85% of M-OCC showed a similar high degree of expansion (P > 0.05), which was displayed by only 25 ± 2% of complexes matured in the presence of LH (P < 0.05 vs. other hormonal treatments). In the absence of gonadotropins, 72 ± 4% of M-OCC showed a low degree of expansion (P < 0.05 vs. other hormonal treatments) (Fig. 2AGo). By the end of culture, the majority of M-oocytes stimulated with both gonadotropins or FSH alone reached the MII stage (95 ± 5 and 89 ± 7%, respectively; P > 0.05), whereas this percentage decreased in the presence of LH (75 ± 9%; P < 0.05 vs. FSH/LH) and without any gonadotropin supplementation (65 ± 6%; P < 0.05 vs. FSH/LH; Fig. 3AGo).


Figure 2
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  		FIG. 2. Analyses of CE degree. The degree of CE was evaluated after 24 h of culture with or without gonadotropin stimulation. A, M-OCC; B, S-OCC cultured alone (IVM); C and D, S-OCC cocultured (Co-IVM) with M-OCC (C) or with M-CC (D). About 80–100 complexes were analyzed for each experimental condition. Data are expressed as the mean ± SEM. Details of statistical analyses are reported in the text.

 

Figure 3
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  		FIG. 3. Gonadotropin effects on oocyte meiotic maturation. S-OCC and M-OCC are cultured alone (IVM) (A), or together (Co-IVM) (B) for 24 h, with or without gonadotropins. Data are presented as the percentages of MII oocytes recorded by the end of culture (mean ± SEM; three independent experiments with five replicates per group per experiment). Significant differences (P < 0.05) are indicated by different letters.

 
Under all the experimental conditions tested, more than 98% of S-OCC displayed a low CE degree (Fig. 2BGo), and only 12 ± 3% of the S-oocytes reached MII (Fig. 3AGo).

CE and meiotic maturation of cocultured M- and S-OCC
About 50% of S-OCC matured in coculture with M-OCC (Fig. 2CGo) or M-CC (Fig. 2DGo) underwent CE with a degree that ranged from moderate to high when stimulated by FSH/LH or FSH alone (P < 0.05 vs. S-OCC cultured under IVM conditions). In contrast, the process of expansion was unaffected by LH as well as by the lack of hormones (P < 0.05 vs. FSH or FSH/LH). When S-OOX were cocultured with M-OCC or M-CC, the degree of CE recorded was not significantly different from that obtained for intact S-OCC (data not shown).

The assessment of meiotic maturation performed at the end of culture revealed that about half of the oocytes obtained from S-complexes (S-oocytes) coincubated with M-OCC or M-CC in the presence of both gonadotropins (FSH/LH) or FSH were able to resume meiosis and to reach MII stage (48 ± 6 and 43 ± 5%, respectively, P > 0.05; Fig. 3BGo). When coculture was performed with LH or without gonadotropins, the percentage of S-oocytes reaching MII was significantly decreased (21 ± 6 and 12 ± 4%, respectively; P < 0.05 vs. FSH/LH).

Time-dependent changes in the nuclear status of M- and S-oocytes cultured under Co-IVM conditions and in the presence of FSH/LH were then analyzed. As control, the same analysis was performed on S-oocytes matured alone (IVM). It was found that at each time point tested (8, 16, and 24 h), a significantly higher percentage of M-oocytes underwent GVBD (74 ± 1%), reached the MI (95 ± 3%), and then the MII (95 ± 3%) stage in comparison with S-oocytes matured under IVM conditions (GVBD, 16 ± 1%; MI, 15 ± 2%; MII, 12 ± 3%; P < 0.05; Fig. 4Go). In addition, the percentages of maturing S-oocytes were higher when maturation was performed under Co-IVM (GVBD, 34 ± 2%; MI, 47 ± 3%; MII, 48 ± 4%) than IVM conditions (Co-IVM vs. IVM, P < 0.05 at each time point tested; Fig. 4Go). Although about 20% of the cocultured S-oocytes resumed meiosis after 24 h of coculture, the percentage of MI-arrested oocytes, i.e. of oocytes showing a partial meiotic competence, was less than 4%. When the percentages of maturation were expressed as the ratio of GVBD/MI and MI/MII, the relative values obtained were comparable. Indeed, for M- and S-oocytes, the ratios of GVBD/MI were 78 and 76% (P > 0.05) and those of MI/MII were 100 and 98%, respectively (P > 0.05).


Figure 4
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  		FIG. 4. Analysis of meiotic stage of sheep oocytes. The nuclear status of M- and S-oocytes under Co-IVM condition and of S-oocytes cultured alone (IVM) was evaluated at 8, 16, and 24 h of culture in the presence of FSH/LH. Results are expressed as the percentage of different nuclear-stage oocytes recorded at each time point (mean ± SEM of five independent experiments). About 100 oocytes were analyzed for each experimental condition. Different letters indicate statistically different values among the experimental groups (P < 0.05).

 
We then evaluated which compartment of M-OCC, somatic or germinal, was able to stimulate meiotic maturation and CE. To this end, S-OCC were cultured in the presence of both gonadotropins with M-OCC, with M-DO, or with their dissociated CC (M-CC) (Fig. 5Go, a–c). After coculture with M-DO, only 10 ± 4% of S-oocytes reached MII stage (Fig. 5bGo), and no CE was detectable (data not shown). By contrast, when cultured with M-OCC (Fig. 5aGo) as well as M-CC (Fig. 5cGo), about 45% of S-oocytes reached MII stage and displayed a degree of expansion ranging from moderate to high (see Figs. 5Go and 2Go, C and D).


Figure 5
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  		FIG. 5. Meiotic maturation of S-oocytes cocultured with intact or dissociated M-complexes. S-OCC (a–c) or S-DO (d–f) were cultured with M-OCC (a and d), M-DO (b), and M-CC (c and e). *, P < 0.05.

 
To determine whether meiosis-inductive signal(s) could target directly the oocytes, S-DO were incubated with M-OCC (Fig. 5dGo) or M-CC (Fig. 5eGo). Results showed that, independently of the different experimental conditions tested, the percentage of S-DO capable of resuming and completing meiotic maturation up to MII was very low and similar to that of S-DO matured alone (Fig. 5Go, d–f).

The positive effect exerted by Co-IVM conditions on meiotic maturation and CE of S-OCC was cell-type dependent. In fact, when S-OCC were cultured with gonadotropin-stimulated mural granulosa cells obtained from M-follicles, a low percentage of the enclosed oocytes reached the MII (11 ± 4%), and no expansion was recorded (data not shown).

Activation of MEK/MAPK in S- and M-OCC
In the next experiments, MEK/MAPK activation was assessed in complexes cultured alone (IVM conditions). When M-OCC underwent maturation in the absence of FSH/LH, CC maintained a basal level of active kinase at any time tested (0, 8, 16, and 24 h); the enclosed oocytes displayed pMAPK after 16 and up to 24 h of culture, in coincidence with MI and MII stage (Fig. 6AGo). By contrast, when IVM was carried out in the presence of FSH/LH, the level of active MAPK increased significantly in CC at 8 h, followed by a sharp decrease to the basal level at 16 and 24 h (Fig. 6BGo). In the corresponding oocytes, the level of MAPK phosphorylation increased at MI (16 h) and further at MII (24 h; Fig. 6BGo). By analyzing MAPK activation in S-OCC, it was evident that gonadotropins did not modify CC/oocyte level of MAPK activity (Fig. 6Go, A and B). In fact, S-CC showed a decrease of kinase phosphorylation after 16 h, whereas the enclosed oocytes never activated this pathway. It was noteworthy that the total amount of MAPK protein did not change at any time tested.


Figure 6
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  		FIG. 6. Western blot analysis of MEK/MAPK activation in IVM OCC showing phosphorylation levels of MEK/MAPK in M- and S-OCC in vitro matured without (A) or with (B) FSH/LH. Samples of oocytes and CC were collected at 0, 8, 16, and 24 h of culture. Each experiment was repeated three times with similar results, and a representative gel is shown.

 
As shown in Fig. 6Go, A and B, MEK activity appeared to be strictly correlated to that of MAPK, according to results obtained in other cell systems (39).

MAPK activation in cocultured M- and S-OCC
To analyze the capacity of paracrine factors to activate the MAPK pathway in cumulus and germ cells of S-complexes, MEK/MAPK activation was analyzed in S-OCC cocultured with (Co-IVM) gonadotropin-stimulated M-OCC at 8, 16, and 24 h of culture (Fig. 7Go). It was found that the oocytes and CC of the M-complexes showed a pattern of kinase activation comparable to that evidenced for IVM conditions (compare Fig. 7AGo with 6B). In contrast, although S-CC evidenced no significant variation of their phosphorylation pattern, S-oocytes displayed at 16 and 24 h the activation of the MAPK pathway (see Fig. 7BGo vs. 6B). Similar results were obtained using M-CC (data not shown). The phosphorylation levels of MEK/MAPK observed in S-DO coincubated with M-OCC or M-CC were always low (data not shown).


Figure 7
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  		FIG. 7. Western blot analysis of MEK/MAPK activation in cocultured (Co-IVM) M-OCC and S-OCC. S-OCC were cocultured (Co-IVM) with M-OCC in the presence of FSH/LH. Phosphorylation levels of MEK/MAPK were assessed in samples of M-oocytes and related CC (A) and S-oocytes and related cumulus (B) collected at 0, 8, 16, and 24 h of coculture. Each experiment was repeated three times with similar results, and a representative gel is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results presented here demonstrate that about 48% of oocytes obtained from small antral follicles (S-oocytes) of prepubertal sheep are able to resume and complete meiotic maturation up to MII, and their surrounding CC (S-CC) to undergo CE when matured in coculture (Co-IVM) with gonadotropin-stimulated OCC or dissociated CC obtained from medium antral follicles (M-OCC and M-CC, respectively). We observed that only a part of the S-oocytes population undergoes both maturational processes. This can be explained by the fact that the population selected for our analysis is composed of oocytes similar in size but showing different degrees of competence.

Gonadotropins are necessary to enable the CC of M-complexes to secrete meiosis-inducing signal(s) that target specifically S-CC and induce the enclosed oocytes to reinitiate and complete the cell cycle. Indeed, by the end of coculture, a low percentage (<4%) of S-oocytes was arrested at MI, a condition indicative of a partial meiotic competence (40). This result is in contrast with that obtained for the pig, because in this species the majority of oocytes with a similar diameter (about 110 µm) remained arrested at the MI stage (41). We also observed that, under Co-IVM conditions, the percentages of M- and S-oocytes undergoing meiotic maturation were different. However, the similar values of the ratio GVBD/MI and MI/MII suggested that meiotic progression occurs with a similar kinetics in both oocyte classes. Taken together, these observations suggest that S-oocytes remain arrested at the GV stage as a consequence of the inability of their surrounding CC to respond to gonadotropin stimulation.

Results from various coculture protocols support the hypothesis that meiosis-inductive signals are delivered to S-oocytes via CC (Fig. 5Go). A different result has been obtained for the mouse. In this species, CC-derived paracrine factor(s) capable of stimulating resumption of meiosis act directly on the DO, even if their positive effects are overridden by the transfer via gap junctions of meiosis-inhibitory molecules (42, 43). This discrepant result can be due either to species-specific differences or to the use of oocytes collected from different classes of antral follicles (preovulatory in the mouse model, small and medium antral in the present model).

It has been recently reported that in the mouse, the oocyte plays a central role in the induction of meiosis resumption and CE. Indeed, Su and colleagues (23) demonstrated that oocyte-derived paracrine factors enable gonadotropin-stimulated companion CC to produce signals stimulating both maturational processes. From our results, a similar model seems not to be applicable to sheep S-oocytes. In fact, regardless the presence/absence of CC, the percentage of S-oocytes reaching the MII stage was not increased by coincubation with M-DO (Fig. 5Go). Moreover, we found that about 50% of S-CC showed a significant improvement of the degree of CE (from low to moderate/high) when incubated with gonadotropin-stimulated M-CC. This doesn’t occur when S-complexes were cultured alone (IVM condition) or together with M-DO. Therefore, unlike the mouse (30), in the sheep CE is regulated by factors produced by somatic cells rather than by the oocyte, thus resembling the porcine model (18, 27, 33, 44, 45). The role of somatic cells is supported also by the finding that CE occurred in S-OOX cultured with M-CC. In the mouse, CE and oocyte developmental competence are regulated by molecules of the TGFβ superfamily, among which are growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (6, 18, 46, 47). Although we cannot rule out the possibility that also under our experimental conditions M- and S-oocytes could release GDF9 or bone morphogenetic protein 15, their concentrations in culture medium might be too low to affect CC and oocytes. Recently, Latham and collaborators (48) proposed that mouse fully grown and growing oocytes secrete different amounts of GDF9 and that fully grown oocytes release factors capable of mitigating the effects of GDF9. A similar modulation might occur for cocultured sheep S- and M-oocytes.

The capacity of M-CC-derived paracrine factor(s) to activate the MEK/MAPK pathway in the somatic and germinal compartments of S-OCC has been then investigated. Due to the lack of literature data, we first examined the pattern of MAPK activation in CC and gametes of M-complexes matured in the absence or presence of gonadotropins. We found that in the sheep, as in other mammals (18, 20, 22, 25, 27, 49), the MAPK cascade is activated during meiotic maturation, first in cumulus and then in the oocyte. Although unstimulated M-CC displayed a basal level of MAPK phosphorylation throughout the whole culture period, gonadotropin supplementation caused a significant rise in the pMAPK level at 8 h of culture, a time corresponding to oocyte GVBD (Fig. 6Go). A similar result has been obtained for FSH-stimulated mouse (23, 49) and pig (21) preovulatory complexes. In the mouse, this activation has been associated with the synthesis of specific somatic proteins and secretion of factors capable of inducing resumption of meiosis (17). On the other hand, the finding that S-CC did not show MAPK activation also after gonadotropin administration strongly supports their immaturity (Fig. 6Go). This could be due to low gonadotropin receptor expression (50, 51), to the absence/inactivation of intermediates of gonadotropin-dependent signal transduction pathways (52, 53), or both. It is also possible that such an immaturity could be due not only to intrinsic differences in CC functional roles but also to differences in the stage of antral follicle development (35).

Concerning germ cells, we found that M-oocytes, independently of culture conditions, showed MEK/MAPK activation at 16 and 24 h of culture, when almost all of them were at MI and MII, respectively. However, our results showed that kinase phosphorylation in germ cells was more efficiently stimulated by hormonal supplementation. This explains the improvement in the percentage of M-oocytes reaching the MII stage. A different result has been reported for porcine oocytes by Liang and collaborators (27). In this species, even if cumulus-enclosed oocytes matured in the absence of gonadotropins, they resumed meiosis later in comparison with stimulated complexes. Despite this, similar levels of MAPK activity were detectable after GVBD regardless of IVM conditions (27). In contrast with M-oocytes, the low percentage of S-oocytes able to reach MII stage never showed an appreciable level of MAPK activation. This occurred also when these oocytes were matured in the presence of gonadotropins (Fig. 6Go). However, we found that all the S-oocytes capable of progressing to MII when cocultured with M-CC showed a pattern of MEK/MAPK phosphorylation similar to that of M-oocytes (Fig. 7Go). These results strongly reinforce our idea that S-oocytes are provided with the molecular machinery controlling resumption of meiosis but that this process can be activated only by appropriate stimuli.

Because in mammalian oocytes meiosis is controlled by maturation-promoting factor activation (25, 49, 54, 55), we can hypothesize that cocultured S-oocytes show a different level of maturation-promoting factor activity/stabilization in comparison with oocytes matured alone. This possibility is currently under investigation.

In sum, our data demonstrate, for the first time, that gonadotropins are necessary to stimulate in sheep M-CC the secretion of factor(s) capable of activating the signaling pathways triggering meiotic maturation and CE in S-complexes. This capacity is not displayed by these complexes because of somatic cell immaturity even after gonadotropin stimulation. These results highlight further that the synchrony between germ and follicle cell development is central for the production of a mature oocyte. Because in recent years many attempts have been made to increase the number of germ cells to be used for in vitro fertilization programs (56, 57), the finding that the immaturity of the somatic compartment impairs the acquisition of oocyte full developmental competence points to Co-IVM procedures as a useful tool to increase the number of potentially fertilizable oocytes.


   Acknowledgments
 
We thank Prof. Rita Canipari for critical reading of the manuscript and helpful suggestions.


   Footnotes
 
Address all requests for reprints to: Prof. Barbara Barboni, Department of Comparative Biomedical Sciences, University of Teramo, 64100 Teramo, Italy. E-mail: bbarboni{at}unite.it.

This research was supported by PRIN 2005 to S.C. and B.B.

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 20, 2007

Abbreviations: CC, Cumulus cells; CE, cumulus expansion; DO, denuded oocytes; GV, germinal vesicle; GVBD, germinal vesicle breakdown; IVM, in vitro maturation; M, medium; MEK, MAPK kinase; MII, metaphase II; OCC, oocyte-cumulus complexes; OOX, oocytectomized complexes; p, phosphorylated; S, small.

Received June 28, 2007.

Accepted for publication September 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tanghe S, Van Soom A, Nauwynck H, Coryn M, de Kruif A 2002 Minireview: Functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Mol Reprod Dev 61:414–424[CrossRef][Medline]
  2. Buccione R, Schroeder AC, Eppig JJ 1990 Interactions between somatic cells and germ cells throughout mammalian oogenesis. Biol Reprod 43:543–547[Abstract]
  3. Cecconi S, Ciccarelli C, Barberi M, Macchiarelli G, Canipari R 2004 Granulosa cell-oocyte interactions. Eur J Obstet Gynecol Reprod Biol 115(Suppl 1):S19–S22
  4. Eppig JJ 2001 Oocyte control of ovarian follicular development and function in mammals. Reproduction 122:829–838[Abstract]
  5. Albertini DF, Combelles CM, Benecchi E, Carabatsos MJ 2001 Cellular basis for paracrine regulation of ovarian follicle development. Reproduction 121:647–653[Abstract]
  6. Gilchrist RB, Ritter LJ, Armstrong DT 2004 Oocyte-somatic cell interactions during follicle development in mammals. Anim Reprod Sci 82–83:431–446
  7. Matzuk MM, Burns KH, Viveiros MM, Eppig JJ 2002 Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science 296:2178–2180[Abstract/Free Full Text]
  8. Bao S, Ushijima H, Hirose A, Aono F, Ono Y, Kono T 2003 Development of bovine oocytes reconstructed with a nucleus from growing stage oocytes after fertilization in vitro. Theriogenology 59:1231–1239[CrossRef][Medline]
  9. Eppig JJ 1996 Coordination of nuclear and cytoplasmic oocyte maturation in eutherian mammals. Reprod Fertil Dev 8:485–489[CrossRef][Medline]
  10. Mattson BA, Albertini DF 1990 Oogenesis: chromatin and microtubule dynamics during meiotic prophase. Mol Reprod Dev 25:374–383[CrossRef][Medline]
  11. Connors SA, Kanatsu-Shinohara M, Schultz RM, Kopf GS 1998 Involvement of the cytoskeleton in the movement of cortical granules during oocyte maturation, and cortical granule anchoring in mouse eggs. Dev Biol 200:103–115[CrossRef][Medline]
  12. Eppig JJ, Viveiros MM, Bivens CM, De La Fuente R 2004 Regulation of mammalian oocyte maturation. Ovary 71:113–11129
  13. Pincus G, Enzmann EV 1935 The comparative behaviour of mammalian eggs in vivo and in vitro. I. The activation of ovarian eggs. J Exp Med 84:655–1054
  14. Mehlmann LM, Jones TL, Jaffe LA 2002 Meiotic arrest in the mouse follicle maintained by a Gs protein in the oocyte. Science 297:1343–1345[Abstract/Free Full Text]
  15. Moor RM, Trounson AO 1977 Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J Reprod Fertil 49:101–109[Abstract/Free Full Text]
  16. Carroll J 2000 Na+-Ca2+ exchange in mouse oocytes: modifications in the regulation of intracellular free Ca2+ during oocyte maturation. J Reprod Fertil 118:337–342[Abstract]
  17. Downs SM, Daniel SA, Eppig JJ 1988 Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic cell origin. J Exp Zool 245:86–96[CrossRef][Medline]
  18. Fan HY, Huo LJ, Chen DY, Schatten H, Sun QY 2004 Protein kinase C and mitogen-activated protein kinase cascade in mouse cumulus cells: cross talk and effect on meiotic resumption of oocyte. Biol Reprod 70:1178–1187[Abstract/Free Full Text]
  19. Mattioli M 1994 Transduction mechanisms for gonadotrophin-induced oocyte maturation in mammals. Zygote 2:347–349[Medline]
  20. Fan HY, Tong C, Lian L, Li SW, Gao WX, Cheng Y, Chen DY, Schatten H, Sun QY 2003 Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization. Biol Reprod 68:968–977[Abstract/Free Full Text]
  21. Ohashi S, Naito K, Sugiura K, Iwamori N, Goto S, Naruoka H, Tojo H 2003 Analyses of mitogen-activated protein kinase function in the maturation of porcine oocytes. Biol Reprod 68:604–609[Abstract/Free Full Text]
  22. Su YQ, Wigglesworth K, Pendola FL, O’Brien MJ, Eppig JJ 2002 Mitogen-activated protein kinase activity in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse. Endocrinology 143:2221–2232[Abstract/Free Full Text]
  23. Su YQ, Denegre JM, Wigglesworth K, Pendola FL, O’Brien MJ, Eppig JJ 2003 Oocyte-dependent activation of mitogen-activated protein kinase (ERK1/2) in cumulus cells is required for the maturation of the mouse oocyte-cumulus cell complex. Dev Biol 263:126–138[CrossRef][Medline]
  24. Tsafriri A, Cao X, Ashkenazi H, Motola S, Popliker M, Pomerantz SH 2005 Resumption of oocyte meiosis in mammals: on models, meiosis activating sterols, steroids and EGF-like factors. Mol Cell Endocrinol 234:37–45[CrossRef][Medline]
  25. Fan HY, Sun QY 2004 Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals. Biol Reprod 70:535–547[Abstract/Free Full Text]
  26. Su YQ, Rubinstein S, Luria A, Lax Y, Breitbart H 2001 Involvement of MEK-mitogen-activated protein kinase pathway in follicle-stimulating hormone-induced but not spontaneous meiotic resumption of mouse oocytes. Biol Reprod 65:358–365[Abstract/Free Full Text]
  27. Liang CG, Huo LJ, Zhong ZS, Chen DY, Schatten H, Sun QY 2005 Cyclic adenosine 3',5'-monophosphate-dependent activation of mitogen-activated protein kinase in cumulus cells is essential for germinal vesicle breakdown of porcine cumulus-enclosed oocytes. Endocrinology 146:4437–4444[Abstract/Free Full Text]
  28. Conti M, Hsieh M, Park JY, Su YQ 2006 Role of the epidermal growth factor network in ovarian follicles. Mol Endocrinol 20:715–723[Abstract/Free Full Text]
  29. Diaz FJ, O’Brien MJ, Wigglesworth K, Eppig JJ 2006 The preantral granulosa cell to cumulus cell transition in the mouse ovary: development of competence to undergo expansion. Dev Biol 299:91–104[CrossRef][Medline]
  30. Vanderhyden BC, Caron PJ, Buccione R, Eppig JJ 1990 Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation. Dev Biol 140:307–317[CrossRef][Medline]
  31. Ochsner SA, Day AJ, Rugg MS, Breyer RM, Gomer RH, Richards JS 2003 Disrupted function of tumor necrosis factor-{alpha}-stimulated gene 6 blocks cumulus cell-oocyte complex expansion. Endocrinology 144:4376–4384[Abstract/Free Full Text]
  32. Ochsner SA, Russell DL, Day AJ, Breyer RM, Richards JS 2003 Decreased expression of tumor necrosis factor-{alpha}-stimulated gene 6 in cumulus cells of the cyclooxygenase-2 and EP2 null mice. Endocrinology 144:1008–1019[Abstract/Free Full Text]
  33. Prochazka R, Nagyova E, Brem G, Schellander K, Motlik J 1998 Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles. Mol Reprod Dev 49:141–149[CrossRef][Medline]
  34. Ledda S, Bogliolo L, Leoni G, Naitana S 1999 Follicular size affects the meiotic competence of in vitro matured prepubertal and adult oocytes in sheep. Reprod Nutr Dev 39:503–508[CrossRef][Medline]
  35. Hunter MG, Robinson RS, Mann GE, Webb R 2004 Endocrine and paracrine control of follicular development and ovulation rate in farm species. Anim Reprod Sci 82–83:461–477
  36. Buccione R, Vanderhyden BC, Caron PJ, Eppig JJ 1990 FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte. Dev Biol 138:16–25[CrossRef][Medline]
  37. Ledda S, Bogliolo L, Leoni G, Naitana S 2001 Cell coupling and maturation-promoting factor activity in in vitro-matured prepubertal and adult sheep oocytes. Biol Reprod 65:247–252[Abstract/Free Full Text]
  38. Lowry Oh, Rosebrough Nj, Farr Al, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275[Free Full Text]
  39. Denhardt DT 1996 Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling. Biochem J 318(Pt 3):729–747
  40. Hampl A, Eppig JJ 1995 Analysis of the mechanism(s) of metaphase I arrest in maturing mouse oocytes. Development 121:925–933[Abstract]
  41. Sedmikova M, Burdova J, Petr J, Etrych M, Rozinek J, Jilek F 2003 Induction and activation of meiosis and subsequent parthenogenetic development of growing pig oocytes using calcium ionophore A23187. Theriogenology 60:1609–1620[CrossRef][Medline]
  42. Downs SM, Mastropolo AM 1994 The participation of energy substrates in the control of meiotic maturation in murine oocytes. Dev Biol 162:154–168[CrossRef][Medline]
  43. Downs SM 2001 A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic induction in isolated mouse oocytes. Zygote 9:71–82[CrossRef][Medline]
  44. Nagyova E, Vanderhyden BC, Prochazka R 2000 Secretion of paracrine factors enabling expansion of cumulus cells is developmentally regulated in pig oocytes. Biol Reprod 63:1149–1156[Abstract/Free Full Text]
  45. Prochazka R, Nagyova E, Rimkevicova Z, Nagai T, Kikuchi K, Motlik J 1991 Lack of effect of oocytectomy on expansion of the porcine cumulus. J Reprod Fertil 93:569–576[Abstract/Free Full Text]
  46. Dragovic RA, Ritter LJ, Schulz SJ, Amato F, Thompson JG, Armstrong DT, Gilchrist RB 2007 Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion. Biol Reprod 76:848–857[Abstract/Free Full Text]
  47. Su YQ, Wu X, O’Brien MJ, Pendola FL, Denegre JN, Matzuk MM, Eppig JJ 2004 Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop. Dev Biol 276:64–73[CrossRef][Medline]
  48. Latham KE, Wigglesworth K, McMenamin M, Eppig JJ 2004 Stage-dependent effects of oocytes and growth differentiation factor 9 on mouse granulosa cell development: advance programming and subsequent control of the transition from preantral secondary follicles to early antral tertiary follicles. Biol Reprod 70:1253–1262[Abstract/Free Full Text]
  49. Abrieu A, Doree M, Fisher D 2001 The interplay between cyclin-B-Cdc2 kinase (MPF) and MAP kinase during maturation of oocytes. J Cell Sci 114:257–267[Abstract]
  50. Calder MD, Caveney AN, Smith LC, Watson AJ 2003 Responsiveness of bovine cumulus-oocyte-complexes (COC) to porcine and recombinant human FSH, and the effect of COC quality on gonadotropin receptor and Cx43 marker gene mRNAs during maturation in vitro. Reprod Biol Endocrinol 1:14[CrossRef][Medline]
  51. van Tol HT, van Eijk MJ, Mummery CL, van den HR, Bevers MM 1996 Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa. Mol Reprod Dev 45:218–224[CrossRef][Medline]
  52. Hoshino Y, Yokoo M, Yoshida N, Sasada H, Matsumoto H, Sato E 2004 Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes. Mol Reprod Dev 69:77–86[CrossRef][Medline]
  53. Xie H, Xia G, Byskov AG, Andersen CY, Bo S, Tao Y 2004 Roles of gonadotropins and meiosis-activating sterols in meiotic resumption of cultured follicle-enclosed mouse oocytes. Mol Cell Endocrinol 218:155–163[CrossRef][Medline]
  54. Marangos P, Verschuren EW, Chen R, Jackson PK, Carroll J 2007 Prophase I arrest and progression to metaphase I in mouse oocytes are controlled by Emi1-dependent regulation of APC(Cdh1). J Cell Biol 176:65–75[Abstract/Free Full Text]
  55. Motlik J, Kubelka M 1990 Cell-cycle aspects of growth and maturation of mammalian oocytes. Mol Reprod Dev 27:366–375[CrossRef][Medline]
  56. Cecconi S, Capacchietti G, Russo V, Berardinelli P, Mattioli M, Barboni B 2004 In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue. Biol Reprod 70:12–17[Abstract/Free Full Text]
  57. Thomas FH, Walters KA, Telfer EE 2003 How to make a good oocyte: an update on in-vitro models to study follicle regulation. Hum Reprod Update 9:541–555[Abstract/Free Full Text]



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