Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation

ELSEVIER

KINETICS OF NUCLEAR MATURATION AND PROTEIN PROFILES OF OOCY'IT_,S FROM PREPUBERTAL AND ADULT CATILE DURING IN VITRO MATURATION H. Khatir, P. Lonergan and P. Mermillod INRA-PRMD, 37380 Nouzilly, France Received for publication: November 24, 1997 Accepted: A p r i l 30, 1998

ABSTRACT The aim of this present study was to compare the kinetics of nuclear maturation between calf and cow oocytes in order to determine if there are differences between the 2 groups which could explain their disparate developmental capacity. The constitutive and neosynthetic protein patterns of cow and calf oocytes and of their corresponding cumulus cells were also compared during in vitro maturation. A total of 397 calf oocytes and 406 cow oocytes was matured in M199 + 10 ng/mL EGF. The first group of oocytes (n=30) was immediately fixed and stained after removal from the follicle, and represent 0 h. The remaining oocytes were removed from the maturation medium at 4, 8, 12, 16, 20 and 24 h respectively. Half were denuded, fixed and stained for nuclear status; while the remainder were radiolabeled with methionine-(35S).Immediately after isolation, all the oocytes were at the GV stage. By 8 h, GVBD had occurred in most oocytes (calf: 97%; cow: 100%) and some had reached pro-metaphase I (calf: 49%; cow: 51%). By 12 h, most of the oocytes were at metaphase I (calf: 84%; cow: 94%). By 16 h, 54% of calf oocytes had reached telophase I or beyond compared with 71% of cow oocytes. This difference between the 2 groups became significantby 20 h, with 89% of cow oocytes (P<0.05) at metaphase II and 71% of calf oocytes. By 24 h of culture, GVBD had occurred in all cases. Most oocytes completed meiosis I and were arrested at metaphase II with the first polar body extruded (calf: 72%; cow: 86%). No differences were noted in the constitutive and the neosynthetic protein profiles of cumulus cells in relation to the age of animal. Changes in neosynthetic protein patterns were observed both in cow and calf cumulus during IVM, and several proteins showed stage-specific synthesis. For the constitutive protein patterns of cow and calf oocytes, there were quantitative (38 and 40 kD) and qualitative (4, 10, 16, 17, 24, 25 and 26 kD) differences between the 2 groups. Only a few differences were observed in neosynthetic proteins between cow and calf oocytes, but there were changes in relation to nuclear status both in cow and calf oocytes. In conclusion, the difference in developmental capacity between cow and calf oocytes may be explained by a difference in the kinetics of nuclear maturation, which was significant at 20 h of culture (with 89% of cow oocytes at metaphase II and 71% of calf oocytes). At the biochemical level, our results indicate that nuclear progression during in vitro maturation of bovine oocytes is linked to changes in protein synthesis by the oocyte itself, while cumulus protein synthesis may either stimulate or modulate the process of oocyte maturation. © 1998by ElsevierScience Inc.

Key words: oocytes, cumulus, nuclear maturation, protein synthesis Acknowledgments The authors thank Dr O. Gerard (OGER) and Dr C. Boccart (LINALUX, Belgium) for providing bull semen; Mr Le Marrehal and the staff of Tours abattoir for providing ovaries; and Professor G. Evans for commenting on an earlier draft. Theriogenology 50:917-929, 1998 © 1998 by Elsevier Science Inc.

O093-691X/98.t$19.00 PII S0093-691X(98)00196-4

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Theriogenology INTRODUCTION

Mammalian oocytes enter meiosis during fetal life and become arrested at late prophase of the first meiotic division (germinal vesicle). Fully grown oocytes reinitiate meiotic maturation in response to gonadotrophins in vivo and, when released from the follicle, in vitro. This involves rupture of the nuclear membrane (GVBD) and progression through metaphase I, anaphase and telophase, with the extrusion of the first polar body and arrest at metaphase II. In addition to these nuclear changes, the oocyte also undergoes cytoplasmic changes necessary for fertilization and subsequent embryonic development (31). These changes involve structural rearrangement of organelles (6, 7, 49) and major changes in protein synthesis patterns (20, 21, 25, 34, 57). Furthermore, concomitant with ooeyte growth and maturation, follicular cells undergo important changes which enable them to permit or inhibit oocyte maturation (58) and to influence the acquisition of developmental competence. Removal of cumulus cells during in vitro maturation, reduces significantly the rate of oocyte maturation, fertilization and embryo development (31, 47, 60). At the biochemical level, marked changes in constitutive (57) and neosynthetic proteins (44, 57) occur in cumulus cells during in vitro maturation. In contrast with mouse and rat oocytes (12, 45, 50), resumption of meiosis in the ooeytes of large domestic animals requires protein synthesis (cattle: 19, 22, 48, 51, 57; sheep: 34; goat: 25; pig: 12). Several authors have examined the pattern of constitutive proteins (23, 27, 57) and those neosynthesised during oocyte maturation (sheep: 54; bovine: 20, 21, 23, 57; goat: 25). However, at present it is not known which proteins are necessary for the acquisition of developmental competence. In cattle, although prepubertal calf oocytes are capable of undergoing nuclear maturation and fertilization at similar rates to cow oocytes, blastocyst yields from such oocytes are significantly reduced compared with that of adult oocytes (8, 11, 23, 27, 39, 41, 46). This deficiency in developmental capacity of oocytes from calves may be due to abnormal cytoplasmic m_zturation of these oocytes. Thus, calf ooeytes represent a good model for elucidating the mechansims which govern the quality of cytoplasmic maturation. In addition, the ability to produce embryos from oocytes recovered from fetal (2) or prepubertal calves (1, 15) offers the potential for markedly reducing the generation interval in cattle, thereby substantially accelerating the rate of genetic gain that can be achieved through embryo transfer. Dominko and First (10) showed that oocytes that extruded the first polar body earliest during maturation were more likely to develop into blastoeysts. Similarly, Van der Westerlaken et al. (53) observed that oocytes that had expelled the polar body at 16 h or 20 h of culture displayed higher momla/blastocyst rates than those without a polar body. These observations indicate that the kinetics of nuclear maturation could be a good indicator of developmental competence acquisition by oocytes. With this background, the aim of the present study was to compare the patterns of constitutive and neosynthetic proteins of calf and cow oocytes and of their corresponding cumulus cells at various time-points during in vitro maturation. We also examined the kinetics of nuclear maturation of calf and cow oocytes in order to determine which factors accounted for the discrepancy in developmental ability of these 2 groups of fully grown ooeytes. MATERIALS AND METHODS Ooeyte Collection and In Vitro Maturation Chemicals were purchased from Sigma Chemical Co (St. Louis, MO, USA) unless otherwise indicated. The details of methods for ooeyte recovery and in vitro maturation (IVM) have been described previously (29). Briefly, cumulus oocyte complexes (COCs) were obtained by aspiration

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of 2 to 6-mm follicles from the ovaries of slaughterhouse calves (4 to 7 mo-old) and cows. Following 4 washes in modified phosphate-buffered saline (PBS, supplemented with 36 p.g/mL pyruvate, 50 I.tg/mL gentamycin and 0.5 mg/mL bovine serum albumin; Sigma, fraction V, cat. # A-9647), groups of up to 50 COCs were transferred to 4-well plates (Nunc, Roskilde, Denmark) containing 500 IxL of maturation medium (see below) for 24 h culture at 39°C in an atmosphere of 5% CO2 in air with maximum humidity. Kinetics of In Vitro Maturation To establish the kinetics of in vitro maturation, COCs from calves (n= 397) and cows (n= 406) were matured in M199+10 ng/mL EGF. A sample of oocytes (n= 30) was fixed and stained as below immediately after removal from the follicle, and represents 0 h. In addition, oocytes were removed from the maturation dishes at 4, 8, 12, 16, 20 and 24 h after the start of culture. Half of the oocytes were denuded by repeated pipetting, fixed in acetic acid:ethanol (h3), stained with orcein and observed under the microscope. Oocytes were classified as germinal vesicle, prometaphase I, metaphase I, anaphase I, telophase I or metaphase II. The remaining were submitted to radiolabeling. Radiolabeling and Electrophoresis The COCs from cow and calf ovaries were either radiolabeled immediately after collection or were matured for 4, 8, 12, 16 and 20 h and subsequently radiolabeled for 3 h. Labeling was performed at 39°C in PBS containing 1 mCi/mL [3sS]-methionine(Express Protein Labelling Mix, NEN) for 3 h. After labeling, the oocytes were denuded of cumulus cells as above and washed 3 times in PBS (without BSA). Cumulus cells were recovered after centrifugation in PBS without BSA and lysed in 100 I.tL of sample buffer (24). Oocytes were lysed in groups of 30 in 15 I.tL of sample buffer (24). Samples were boiled for 3 min and stored at -20°C until electrophoresis. The incorporation of [35S]-methionine into cumulus cells was determined by liquid scintillation. Equivalent cpm per sample were analyzed. A minimum of 3 replicates was performed for each experiment to ensure reproducibility. Thawed samples were centrifuged (13,000 x g for 5 rain), and the radiolabeled proteins were resolved by SDS-PAGE on homogenous slab gels (12% of acrylamide; 4% of bis-acrylamide). Groups of 30 oocytes and their corresponding cumulus cells were used for analysis of constitutive and neosynthetic proteins. Proteins of known molecular weight range (35 to 190 kD, Sigma cat # SDS 7B) were run simultaneously as standards. To observe the constitutive proteins, gels were silver-stained using a protocol modified from Morrissey (35). Briefly, gels were prefixed in a solution of 40% methanol (v/v) and 10% acetic acid (v/v) in 50% water overnight and then fixed for 30 min in 10% glutaraldehyde (v/v). They were then washed 5 times (20 min) in water and immersed in 5 I.tg/mL dithiothreitoi (DTE) for 30 min. They were subsequently treated with 0.1% silver nitrate (w/v) for 30 min, rinsed rapidly with water, and developed (solution of 0.3 M sodium carbonate containing 0.0135% formaldehyde). Staining was stopped by washing in 10% acetic acid (v/v). Finally, the gels were photographed. For analysis of neosythesized proteins, the gels were treated with Amplify (Amersham) for 30 min, dried, and exposed for 10 d (Hyperfilm-MP, Amersham) at -70°C. Statistical Analysis Data of the kinetics study were analyzed by Chi-square test. The level of significance was P<0.05.

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RESULTS Kinetics of IVM Results of the kinetics of in vitro maturation of calf and cow oocytes arc shown in Figure 1. Oocytes, isolated from follicles and immediately fixed, contained a GV in all cases (100%). The germinal vesicle breakdown (GVBD) occurred in most oocytes between 4 and 8 h of culture. By 8 h, GVBD had occurred in most oocytes (calf: 63165, 97%; cow: 72/72, 100%), and some oocytes had reached pro-metaphase I (calf: 32/65, 49%; cow: 37/72, 51%). At 12 h, most of the oocytes were at metaphase I (calf: 45153, 84%; cow: 69/73, 94%). It was also at 12 h that the first signs of cumulus expansion became apparent. There was a discrepancy between the 2 groups by 16 h as only 54% of the calf oocytes (34162) had reached tclophasc or beyond compared with 71% of the cow oocytcs (51/72). This difference between the 2 groups became significant by 20 h as 89% of the cow oocytes (58165; P<0.05) were at metaphase II compared with 71% of the calf oocytes (49169). 100

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Figure 1. Kinetics of nuclear maturation of cow and calf oocytes matured in M199 + 10 ng/mL EGF. The asterisk indicates that the difference is significant (p<0.05). Oocytes were classified as germinal vesicle (GV), pro-metaphase I (PMI), metaphase I (MI), anaphase I (AI), telophase I (TI) or metaphase 1I (MII).

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921

Following culture for 24 h, GVBD occurred in all cases (calf: 78/78, 100%; cow: 59/59, 100%). Most coeytes had completed meiosis I and were arrested at metaphase II with the first polar body extruded (calf: 56/78, 72%; cow: 51/59, 86%). Oecyte and Cumulus Cell Protein Profiles Protein m'offle of calf and cow cumulus cells durin~ IVM. Profile results are shown in Figure 2. No differefices were noted in the constitutive protein-profiles between cow and calf cumulus cells during IVM. The molecular weights of the major proteins were 18, 30, 40, 45, 52, 62, 76, 80, 92 and 106 kD (Figure 2). kD

106~ 92---4162--I~ 52-1~ 45-t~ 40--~

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18~ 0

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8 Cow

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Times (hours)

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Calf

Figure 2. Constitutive proteins profiles of cow and calf cumulus cells at different times (in hours) of in vitro maturation in M199 + EGF. Each lane represents a sample of the cumulus proteins pooled from 30 cumulus-ooeyte complexes. Gels were silver stained. Protein nrofile of calf and cow oocvtes durin~ IVM. Profile results are shown in Figure 3. Comparison of protein profiles of oocytes from cows and calves during IVM showed quantitative and qualitative variations related with age/pubertal status of animal (i.e., cow vs calf). Differences related with the age of animal in the evolution of proteins profiles were characterized by an increase in the intensity of certain bands (38 and 40 kD) between 0 and 20 h in cow compared with calf ooeytes. Bands 4, 10, 16, 17, 24, 25 and 26 kD were present at all times

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examined during 1VM of cow oocytes but were absent from calf oocytes except at 20 h when protein bandes 4, 10, 24, 25 and 26 kD appeared. kD

42

30 26-.--I1~ 25/24'"

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Figure 3. Constitutive proteins profiles of cow and calf ooeytes, free of cumulus cells, at different times (in hours) of in vitro maturation in M199 + EGF. Each lane represents 30 oocytes from which cumulus cells were removed prior to analysis. Gel was silver stained. Protein neosynthesised by calf and cow cumulus cells durin~ IVM. Protein results are shown in Figure 4. No differences in cumulus cell protein profiles were observed between adult and prepubertal oocytes. However, quantitative and qualitative changes were noted in both cow and calf cumulus cells during IVM. These changes were characterized by an increase in the intensity of certain bands after 4 h of culture (172, 97 and 74 kD). The bands of 112 and 97 kD, which were synthesized at high levels during the early stages of IVM (0 to 8 h of culture), decreased in intensity between 8 and 20 h. The intensity of a 55 kD protein showed a slight decrease after 8 h of culture. In contrast, a 67 kD protein showed a gradual increase in synthesis after the initiation of IVM (4 to 20 h). Bands 90 and 29 kD, present before maturation (0 h), were absent during maturation (4 to 20 h of culture), at which time bands 258, 87, 56 and 28 kD were observed and persisted between 4 and 20 h of maturation. The synthesis of proteins of 180 and 172 kD ceased during the later stage of maturation. They were present at 0 to 2 h and disappeared at 16 to 20 h. Proteins of 72 and 74 kD were present only during the first 4 h of culture. A band of 94 kD, absent before IVM, appeared

Thedogeno~gy

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between 4 and 8 h of culture and disappeared afterwards. All these changes were similar for calf and cow cumulus cells. kD 258--I1~ 180--I~ 172~ 112~

~

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Figure 4. Neosynthesis protein profiles of cow and calf cumulus cells at different times (in hours) of in vitro maturation in M199 + EGF. Each lane represents a sample of the cumulus proteins pooled from 30 cumulus-oocyte complexes. Gels were exposed to Hyperfilm to detect radiolabeled methionine incorporation. Protein neosynthesised by calf and cow oocytes during IVM. The profiles of proteins newly synthesized by cow and calf oocytes are presented in Figure 5. Only a few differences were noted in the neosynthetic protein profiles in relation to the age of animal. For example, before maturation (0 h), the band of 28 kD was more intense in cow compared to calf oocytes. However, quantitative and qualitative changes were noted in both cow and calf oocytes during IVM. For simplicity, we restricted the present study only to bands showing marked changes. These changes were characterized in cow and calf oocytes by a decrease in intensity of a 45 kD band between 12 and 20 h of culture. The band of 70 kD, present before IVM, increased in

The~ogenology

924

intensity after the initiation of culture (0 to 4h), decreased at 8 h and completely disappeared thereafter. However, the protein of 67 kD showed a slight increase in synthesis at 16 h and a 39 kD protein showed a gradual increase in intensity between 4 and 16 h of IVM and decreased after 20 h. The synthesis of certain proteins ceased during maturation, namely the band of 52 kD, which was present only before maturation (0 h) and disappeared after 4 h, and the band of 28 kD, which was observed only during the first 8 h of culture. The bands of 49 and 48 kD, absent before IVM, appeared between 4 and 20 h of culture along with a slight decrease in synthesis of a 48 kD band at the end of culture.

kD

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: ~ : 49 48

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Figure 5. Neosynthesis protein profiles of cow and calf oocytes at different times (in hours) of in vitro maturation in M199 + EGF. Cumulus-oocyte complexes were labeled during 3 hours. Each lane represents 30 oocytes. Gels were exposed to Hyperfilm to detect radiolabeled methionine incorporation.

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DISCUSSION The kinetics of IVM reported here are comparable to those previously reported for cattle (9, 48), sheep (34) and goats (25). The influence of the composition of the maturation medium on this kinetics has been previously shown (30). Cow oocytes, matured in M199 alone, reach metaphase II at a lower rate than those matured in the presence of fetal calf serum (FCS), and the difference in kinetics is already apparent by 12 h of culture. In the presence of FCS, 93% of the oocytes had reached metaphase I compared with 60% for M199 alone (30). This diserepeney between the 2 media was obvious for the remainder of the culture period, with 84% of the oocytes reaching metaphase II by 20 h in M199+FCS compared with 53% in M199 alone. Following 24 h of culture, 18% of the oocytes in M199 remained at GV compared with only 1% in the presence of FCS. We have demonstrated in this study a difference in the kinetics of 1VM in the same maturation medium (M199+EGF) for 2 groups of ooeytes from the same species. Again, this difference may translate into a difference in blastocyst rates further along the developmental axis. These conditions have routinely resulted in 90 to 95% nuclear maturation and 80 to 85% fertilization for both cow and calf oocytes. However, in earlier studies we obtained only 15 to 20% blastocysts for calf oocytes and 40 to 45% for adult oocytes following in vitro fertilization and embryo culture (5, 23, 29). Such kinetics may have implications for the outcome of IVF. Various reports in recent years point to the fact that it is the fastest developing embryos in vitro which are most likely to be comparable to their in vivo counterparts. Oocytes that extrude the first ~3olar body early during maturation are likely to develop into blastocysts (10, 53). Moreover, the earliest cleaving zygotes after IVF (within 40 h) are more likely to develop to blastocysts than those that begin to cleave later (17, 28). In our present study we compared the kinetics of nuclear maturation of cow and calf ooeytes in order to see if there were any differences which could explain the discrepancy in the developmental ability of these 2 groups of oocytes. In general and at different stages of IVM, meiotic progression of calf oocytes was delayed compared with that of cow oocytes. This difference between the 2 groups of oocytes became significant by 20 h. After 24 h of culture, more calf than cow oocytes were arrested before metaphase II. The difference between cow and calf oocytes in terms of developmental capacity may be explained by a difference in the kinetics of nuclear maturation of these oocytes. Mammalian oocytes have been divided into 2 broad groups based on their dependence on protein synthesis for resumption of meiosis (GVBD). In mouse and rat oocytes, GVBD and chromatin condensation occur in the presence of protein synthesis inhibitors (12, 45, 55), but meiotic spindle assembly is impaired (18). In contrast, oocytes from sheep, pigs and cattle fail to undergo GVBD under similar conditions (13, 19, 30, 34), although they may exhibit partial chromatin condensation (36). In vitro maturation of bovine oocytes depends upon the synthesis of several distinct and temporally expressed proteins that may play roles in the highly ordered sequence of events that culminate in oocyte maturation (57). In conjunction with GVBD, which occurs between 6 and 8 h after the beginning of culture (51), marked changes in protein synthesis and phosphorylation were observed in the bovine oocyte (21). The present study demonstrates that marked changes occur in the protein profiles of bovine oocytes and their cumulus cells during IVM, and that several proteins manifested stage-specific synthesis in both oocytes and cumulus cells. In fact, Sirard et al. (48) demonstrated that immature COCs have 4 phases of protein synthesis during a 24 h IVM period; protein synthesis is required for GVBD, for progression to MI, and then to MII, and finally to maintain MII. In our study, the comparison of cow and calf oocyte protein profiles during IVM showed quantitative (38 and 40 kD) and qualitative (4, 10, 16, 17, 24, 25 and 26 kD) differences between the 2 groups from 0 to 16 h of culture. Similar to the findings of IAvesque and Sirard (27), our

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Theriogenology

results indicate that these differences in protein between cow and calf oocytes may be associated with their developmental capacity. However, these differences were not observed at 20 h. Concerning neosynthetic protein profiles, only few differences were observed between cow and calf oocytes. However, as shown by others for adult oocytes (21, 34, 57), protein neosynthesis profiles are continually changing during meiosis in both cow and calves oocytes. In fact, some bands were present only at the GV stage, (e.g., the bands of 52 and 70 kD and disappeared afterwards). Other proteins appeared after initiation of culture in presence of EGF (48 kD) in both adult and prebubertal oocytes. We have previously demonstrated that the addition of 10 ng/mL EGF during in vitro maturation of bovine oocytes increased the synthesis of a 48 kD band at the end of maturation in calf (23) and in cow oocytes (29). In the present study, this band of 48 kD, which was absent before IVM, appeared between 4 and 20 h of culture, with a slight decrease in synthesis after 20 h of culture. The 48 kD protein observed here may be a cyclin-B, which in association with dephosphorylated p34 cdc2 represents the active form of meiosis promoting factor (MPF) and consequently, permits the resumption of meiosis in oocytes. A 45 kD protein decreased in cow and calf oocytes at the end of maturation. It has been identified by others as actin, one of the major synthetic products of the immature oocyte (44). Moor et al (33). reported that this protein accounted for 8 to 10% of the total synthesized proteins in the ovine oocyte. The decrease in actin synthesis would seem to be an essential characteristic of a normal maturation process (25). The synthesis of the 45 kD band was also greatly reduced after removal of the cumulus cells (37). Another protein, a 39 kD band, showed a gradual increase of synthesis in cow and calf oocytes between 8 and 20 h of IVM, confirming the results of Wu et al. (57). Using Western blot and/or immunoprecipitation analysis, a band of 39 kD was previously detected as a c-mos protein in mature bovine oocytes (52, 56, 57). In their study, Wu et al. (57), suggested that this protein of 39 kD may be equivalent to the product of protooncogene c-mos (p39 mos) that is known to act as MPF stabilizer in mouse and xenopus oocytes, at the end of meiosis (38, 42, 59). Recently, it was demonstrated in the xenopus oocyte that c-mos may also play a role in the resumption of meiosis (see 14 for review). The band of 28 kD, which was present in adult and prepubertal oocytes only between 0 and 8 h of culture, may be involved in the transition from GVBD to the MI stage, whereas the band of 67 kD, which increased in synthesis at 16 h of culture, may play a role in the entry to MH. In the follicle, an oocyte has a close and important relationship to its surrounding somatic cells during its growth and maturation. Several studies have demonstrated that removal of cumulus cells during in vitro maturation, significantly reduces the rate of oocyte maturation, fertilization and embryo development (31, 47, 60). At the biochemical level and in parallel with the changes in oocyte's protein profiles, marked changes in constitutive (57) and neosynthetic proteins (44, 57) occur in cumulus cells during in vitro maturation. In our study, we compared the cumulus cells protein patterns between cow and calf oocytes and related them to the oocyte's nuclear status. No differences were noted in the constitutive and the neosynthetic protein profiles between adult and prepubertai cumulus cells. However, changes in neosynthetic protein patterns were observed in both cow and calf cumulus cells during IVM, and several proteins showed stage-specific synthesis. Similarly to the of Wu et al. (57), we observed that there is a difference in protein species between oocytes and cumulus cells. This difference may be explained by the difference in function, in metabolism, and in morphology of these 2 types of cell. However, the evolution of oocytes and cumulus protein patterns together with nuclear status confirm the important correlation that exists between the oocyte and its surrounding somatic cells during oocyte maturation (3, 57). In fact, in the follicle, granulosa cells, cumulus cells and the oocyte were found to be metabolically coupled via gap junctions which provides physical pathways for intercellular communication (40) and metabolite transfer (16). In addition, the oocyte may also regulate cumulus cell expansion by secreting a specific factor (s) (4) or by stimulating cumulus-hyaluronic acid synthesis (43). In addition, some proteins synthesized by the cumulus cells appear to trigger or inhibit oocyte growth and maturation (58).

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In conclusion, the difference between cow and calf oocytes in terms of developmental capacity may be explained, at least in part, by a difference in the kinetics of nuclear maturation, which was significant at 20 h of culture (with 89% of cow oocytes and 71% of calf oocytes at MII). From a biochemical point of view, we observed differences in protein profiles between cow and calf oocytes which could possibly explain the difference in their developmental competence. No differences were noted in cumulus cell proteins in relation to the age of animal. However, changes in neosynthetic protein patterns were observed in both cow and calf oocytes and in their corresponding cumulus cells during IVM, and several proteins showed stage-specific synthesis. This indicated that the nuclear progression during in vitro maturation of the bovine oocyte is linked to changes in protein synthesis by the oocyte itself, while cumulus protein may either stimulate or modulate the process of oocyte maturation. REFERENCES 1. Armstrong DT, Holm P, Irvine B, Petersen BA, Stubbings RB, McLean D, Stevens GF, Seamark RF. Laparoscopic aspiration and in vitro maturation of oocytes from calves. Tberiogenology 1991; 35 (Suppl 1): 182 ahstr. 2. Betteridge KJ, Smith C, Stubhings RB, Xu KP, King WA. Potential genetic improvement of cattle by fertilization of fetal oocytes in vitro. J Reprod Fertil 1989; 38 (Suppl): 87-98. 3. Buccione R, Schroeder AC, Eppig JJ. Interaction between somatic cells and germ cells throughout mammalian oogenesis. Biol Reprod 1990a; 43: 543-547. 4. Buccione R, Vanderhyden BC, Caron PJ, Eppig JJ. FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor (s) secreted by the oocyte. Dev Biol 1990b; 138: 16-25. 5. Carolan C, Lonergan P, Mermillod P. Factor affecting bovine embryo development in syntheic oviduct fluid following oocyte maturation and fertlization in vitro. Theriogenology 1995; 43: 1115-1128. 6. Cran DG. Qualitative and quantitative structural changes during pig oocyte maturation. J Reprod Fertil 1985; 74: 237-245. 7. Cran DG, Cheng WTK. Changes in cortical granules during porcine oocyte maturation. Gamete Res 1986; 11: 311-319. 8. Damiani P, Fissore RA, Cibelli JB, Long CR, Balise JJ, Robl, JM, Duby RT. Evaluation of developmental competence, nuclear and ooplasmic maturation of calf oocytes. Mol Reprod Dev 1996; 45: 521-534. 9. De Loos F, Zeinstra E, Bevers MM. Follicular wall maintains meiotic arrest in bovine oocyte cultured in vitro. Mol Reprod I ~ v 1994; 39: 162-165. 10. Dominko T, First NL. Kinetic of bovine oocytes maturation allows selection for development competence and is affected by gonadotropins. Tberiogenology 1992, 37:203 abstr. 11. Duby RT, Damiani P, Looney CR, Fissore RA, Robl JM. Prepubertal calves as oocyte donors: promises and problems. Theriogenology 1996; 45:121-131. 12. Ekholm C, Magnusson C. Rat oocyte maturation: Effects of protein synthesis inhihitors. Biol Reprod 1979; 21: 1287-1293. 13. Fuika J Jr, Motlik J, Fulka J, Jilek Fet. Effect of cyclobeximide on nuclear maturation of pig and mouse oocytes. J Reprod Fertil 1986; 77: 281-285. 14. Gebauer F, Richter JD. Synthesis and function of mos: the control switch of vertebrate oocyte meiosis. Bio Essays 1997; 19: 23-27. 15. Georges M, Massey JM. Velogenetics, or the synergistic use of marker assisted selection and germ-line manipulation. Theriogenology 1991; 35:151-159. 16. Gilula NB, Epstein ML, Beers WH. Cell to cell communication and ovulation: a study of the cumulus-oocytes complex. J Cell Biol 1978; 78: 58-75. 17. Grisart B, Massip A, Dessy F. Cinematographic analysis of bovine embryo development in serum-free oviduct-conditioned medium. J Reprod Fertil 1994; 101: 257-264. 18. Hashimoto N, Kishimoto T. Regulation of meiotic metaphase by a cytoplasmic maturationpromoting factor during mouse oocyte maturation. Dev Biol 1988; 126: 242-252.

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