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Biology of Reproduction 65, 247-252 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Cell Coupling and Maturation-Promoting Factor Activity in In Vitro-Matured Prepubertal and Adult Sheep Oocytes1

S. Ledda,a, L. Boglioloa, G. Leonia, and S. Naitanaa

a Department of Animal Biology, University of Sassari, 07100 Sassari, Italy

ABSTRACT

We examined some differences between prepubertal and adult ovine oocytes; in particular we analyzed the functional status of the cumulus-oocyte complex, protein synthesis during in vitro maturation, and because no information is available on prepubertal and adult sheep, maturation-promoting factor (MPF) fluctuations throughout meiotic progression both in prepubertal and adult sheep oocytes. After 24 h of maturation, percentages of MII oocytes were similar between prepubertal and adult animals. Electron microscopy examinations showed that prepubertal oocytes had fewer transzonal projections than adult oocytes. Methionine uptake was significantly lower in prepubertal cumulus-enclosed oocytes examined through meiotic progression. On the contrary, denuded prepubertal oocytes showed a higher methionine incorporation in the first 4 h of incubation compared with adult oocytes. We also found some differences in MPF activity between prepubertal and adult oocytes at MII stage. In fact, prepubertal MII oocytes had a significantly lower level of MPF activity than adult oocytes did and, after fusion with germinal vesicle oocytes, they were unable to induce nuclear breakdown and chromosome condensation 1–2 h post-fusion, whereas adult MII oocytes could induce these processes. Our findings show that the lesser competence of prepubertal oocytes could be due to morphological anomalies and alterations in physiological activity and that oocytes do not reach full developmental competence until puberty.

kinases, oocyte development

INTRODUCTION

During the early neonatal period, the ovine ovary shows morphological changes with a 7- and 11-fold increase in weight between birth and 8 wk of age, followed by a decline up to puberty [1]. At this time oocytes are arrested at the dictyate stage of first meiotic prophase, and only after stimulation of the gonadotropins and local growth factors do they acquire the ability to progress to meiotic maturation with germinal vesicle breakdown (GVBD), formation of the first meiotic spindle, expulsion of the first polar body, and arrest at metaphase of the second meiotic division until sperm penetration [2]. Full meiotic maturation can also occur when, after removal from the follicles, oocytes are cultured in vitro coupled with cumulus cells [3], which permit nutritional support and transport of messenger molecules from follicle cells to the oocyte [4, 5]. The occurrence of these events is accompanied by changes in the phosphorylation patterns of various cellular proteins [6, 7] and structural modification [8, 9]. Meiotic progression is regulated by the activity of the maturation-promoting factor (MPF), the universal cell cycle regulator of both mitosis and meiosis. This factor is a serine/threonine protein kinase composed of a catalytic subunit, cyclin-dependent kinase 1, and a regulatory subunit, cyclin B [10, 11]. MPF activity is controlled by the association of cdk1 with cyclin B2, forming pre-MPF, which is subsequently activated by specific dephosphorylation of cdk1 by the homologue of the yeast phosphatase, cdc25 [12, 13]. MPF activity is usually determined in vitro by phosphorylation of exogenous histone H1 used as a substrate [14]. Changes in histone H1 kinase activity have been analyzed during maturation of mouse [15], pig [16], rabbit [17], goat [18], bovine [19], and horse [20] oocytes.

At present, several studies on prepubertal animals have reported the birth of live offspring from bovine [21–23] and ovine [24–26 ] donors. However, in the mouse [27], bovine [28], goat [29], pig [30], and sheep [31] developmental competence of in vitro-matured oocytes from prepubertal animals has been reported to be lower than that of oocytes derived from adult animals [32]. Moreover, in vitro-matured oocytes of prepubertal bovine showed some defective aspects [33–35] such as abnormal chromatin microtubule configurations and protein synthesis. An increase in parthenogenetic activation and polyspermic penetration has also been observed in lamb oocytes compared with those derived from adult animals [31, 36].

On the basis of these observations the aim of the present study was to analyze some differences between prepubertal and adult ovine oocytes; in particular the functional status of the cumulus-oocyte complex (COC), protein synthesis during in vitro maturation, and because no information is available on prepubertal and adult sheep, MPF fluctuations throughout meiotic progression were studied.

MATERIALS AND METHODS

Chemicals

All chemicals in this study were purchased from Sigma Chemical Company (St. Louis, MO) unless stated otherwise.

Recovery and Culture of Oocytes

Ovaries from juvenile (30–40 days of age, body weight 6–12 kg) and adult sheep were collected at local slaughterhouses and transported to the laboratory at room temperature within 1–2 h in Dulbecco PBS with antibiotics. After three washes in fresh PBS with penicillin and streptomycin, ovaries were sliced using a microblade and the contents released in sterile Petri dishes containing Hepes-buffered TCM 199 supplemented with 0.1% (w/v) polyvinyl alcohol (PVA) and antibiotics. COCs of prepubertal and adult animals were selected according to previously defined criteria [3]. COCs with several intact cumulus cell layers (ranging between 5 and 20 granulosa cell layers) and a homogeneous cytoplasm were chosen for in vitro maturation. In addition, COCs from prepubertal ovaries were selected on the basis of their dimension using a calibrated eyepiece of an inverted microscope, and only oocytes with an outside diameter (zona pellucida included) >135 µm were used. For each ovary, the number of oocytes with the morphology described above was recorded.

Maturation of Oocytes

After several washes in Hepes-buffered TCM 199, prepubertal and adult sheep COCs were cultured in 35-mm Petri dishes (50 oocytes per dish) using 2 ml of TCM 199 modified with 10% fetal calf serum (FCS), 0.1 UI/ml FSH, and 0.1 UI/ml LH (Pergonal; Serono, Rome, Italy) at 39°C in a humidified environment of 5%CO2 in air. Three replicates were performed to compare the speed of meiotic progression between groups of prepubertal (n = 360) and adult (n = 360) oocytes.

To evaluate the meiotic progression at different culture times (0, 8, 14, and 24 h) cumulus cells were removed by pipetting cumulus-enclosed oocytes through a small-bore glass pipette; thereafter oocytes were stained with 1 µg/ml Hoechst 33342 in TCM 199 for 15 min and were assessed under an inverted microscope (Diaphot, Nikon). The rate of oocytes at germinal vesicle (GV), GVBD, metaphase I (MI), and metaphase II (MII) stages was recorded.

Electron Microscopy

Thirteen oocytes from prepubertal sheep and 12 from adult sheep were processed for transmission electronic microscopy (TEM) immediately after collection from the ovarian follicles. After collection from the ovaries, prepubertal and adult cumulus-enclosed oocytes were extensively washed in PBS and fixed for 60 min in 2.5% glutaraldehyde and 0.75% paraformaldehyde in PBS. They were washed briefly in two changes of 0.1 M cacodylate buffer and postfixed for 60 min in 2% osmium tetroxide, and washed in distilled solution (0.5%) overnight at 4°C. Fixed oocytes were then dehydrated through increasing concentrations of acetone and embedded in Epon 812. To minimize bias due to the tangential sectioning, transects were made at the midpoint of the oocytes. Thereafter the sections were contrasted with uranyl acetate and lead citrate, and examined using a transmission electron microscope. Eight oocytes from lamb ovaries and seven from adult ovaries were sectioned and examined for the foot processes through the zona pellucida.

Protein Labeling and Analysis

This experiment was performed to analyze protein synthesis during the first and the last 4 h of meiotic maturation in COCs and denuded oocytes from prepubertal and adult sheep.

Groups of 5–15 cumulus complexes (n = 6) and denuded oocytes (n = 6) from both prepubertal and adult animals were collected at 0 and 20 h from the maturation system and cultured separately for protein labeling in 50-µl microdrops of methionine-free minimum essential medium (MEM) containing 35S-methionine and 35S-cysteine (Trans 35S label; ICN Pharmaceuticals, Costa Mesa, CA; specific activity >1000 mCi/mmol) at a concentration of 0.5 mCi/ml. Oocytes were cultured for 4 h at 39°C in 5% CO2 in air. After culture, cumulus-enclosed oocytes were denuded from granulosa cells with a glass pipette as described above. Oocytes from both groups were washed twice in TCM 199 plus 0.1% PVA, and assessed for nuclear stage. Oocytes in GV and MII stage (labeled at 0–4 and 20–24 h of culture, respectively) were collected (5 oocytes/vial) in 10 µl of storage buffer (1.87 mg/ml EDTA, 0.42 mg/ml NaF, and 1.84 mg/ml NaVO4 in PBS) and stored at -80°C until analysis.

After thawing, samples of labeled oocytes were precipitated with 20% trichloroacetic acid (TCA) and counted in a Tricarb ß-counter (Packard, Canberra, Australia) to evaluate quantitative incorporation of labeled amino acids into proteins.

Protein analysis was performed according to the method of Laemmli [36]. Stored, frozen oocytes were lysed in SDS sample buffer and submitted to one-dimensional SDS-PAGE in a 12% running gel. After electrophoresis, gels were washed in water for 30 min and submitted to fluorography according to the method of Chamberlain [37]. The gels were dried at 70°C for 1.5 h in a gel drier (Bio-Rad, Hercules, CA) and were subsequently exposed at -70°C to ß-max x-ray film (Amersham, Buckinghamshire, U.K.). The analysis was repeated three times for each group and during the two different culture times.

Histone H1 Kinase Assay

At 0, 4, 12, 20, and 24 h during maturation, oocytes from prepubertal and adult ovine were stripped of their granulosa cells by pipetting through a narrow-bore pipette and were thereafter briefly evaluated by Hoechst staining. Groups of five similarly staged oocytes were placed in 2 µl of ice-cold collection buffer (PBS supplemented with 1 mg/ml PVA, 5 mM EDTA, 10 mM Na3VO4, and 10 nM NaF) and then stored at -70°C pending MPF assays.

Histone H1 kinase was assayed as described by Naito and Toyoda [38] with some modifications. After thawing, samples were brought to a final volume of 9 µl with a solution containing 45 mM ß-glycerophosphate, 12 mM p-nitrophenilphosphate, 20 mM (3-N-morpholino ß-propanesulfonic acid MOPS-KOH) pH 7.2, 12 mM MgCl2, 12 mM ethylenegluco-bis (ß-aminoethyl ether) N,N,N',N'-tetraacetic-acid EGTA, 0.1% mM EDTA, 0.8% mM dithiothreitol (DTT), 2.3 mM NaVO4, 2 mM NaF, 0.8 mM PMSF, 15 µg/ml leupeptin, 30 µg/ml aprotinin, 1 mg/ml PVA, 1 mg/ml histone H1 (type III-S from calf thymus, Sigma), 2.2 µM protein kinase inhibitor peptide (Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asu-Ala-Ile-His-Asp), and 1.8 MBq/ml {gamma}-[32P]ATP (166.5 TBq/mmol, ICN Pharmaceuticals, Costa Mesa, CA).

The reaction started after the addition of {gamma}-[32P]ATP and was performed for 30 min at 37°C. The assay was stopped in 2x concentrated SDS sample buffer and boiled for 5 min. Proteins were separated on 1-dimensional SDS-PAGE electrophoresis as described by Laemmli [36]. After electrophoresis the gels were dried and exposed to Amersham ß-max x-ray films at -70°C. Three replicates were performed to asses H1 kinase activity of prepubertal and adult oocytes at different culture times. The activity of MPF was quantified by measuring the density of the bands in the autoradiographic film with a densitometer. The activity of every sample at each point was measured and an average measure was determined. We arbitrarily designated the activity in MI oocytes to be 100% and the others as being a proportion of this activity.

Bioactivity of MPF Using Oocyte-Fusion Experiments

Procedures for oocyte fusion were described in detail by Fulka et al. [39]. Briefly, the zone pellucidae were dissolved by pronase treatment (0.1%) and the MII stage oocytes from prepubertal and adult animals were agglutinated with oocytes at GV stage by incubation in PBS with phytohemagglutinin (300 µg/mg). Careful sucking of the pairs of oocytes into a very narrow pipette ensured the required close membrane contact. The oocyte pairs were then transferred in a fusion chamber containing an isotonic solution of glucose (5%) to be fused by applying one direct-current pulse (80 µsec of 1.25 kV/cm). The pairs of oocytes were washed several times in TCM 199 supplemented with 10% fetal calf serum (FCS) and cultured in the same medium. One hour after the induction of fusion the fused and unfused oocytes were fixed in acetic acid-alcohol (1:3), stained with aceto orcein, and evaluated under phase contrast microscopy.

Statistics

Chi-square analysis was used to analyze fusion data, and ANOVA was performed for both methionine incorporation and H1 kinase data. Data were considered statistically different at P < 0.05.

RESULTS

Oocyte Recovery and Maturation

The mean number of oocytes collected from a single ovary was significantly lower (P < 0.001) in adult sheep (mean ± SD; 8.4 ± 4), compared with prepubertal sheep (35.8 ± 12). During 24 h of culture the meiotic progression time and the maturation rate were similar in oocytes derived from both prepubertal and adult sheep. In fact, results of four replicates showed that there were no differences in the percentages of GVBD (82.8% vs. 85.1%), metaphase I (89.1% vs. 87.2%), and metaphase II (83.2% vs. 87.5%) when evaluated after 8, 14, and 24 h of maturation, respectively, between oocytes from prepubertal and adult sheep.

Electron Microscopy Examination

The morphology of cytoplasmic organelles, namely mitochondria, lipid droplets, Golgi complexes, and endoplasmic reticula at GV stage were similar for oocytes obtained both from prepubertal and adult sheep. A large proportion of mitochondria, existing as aggregates dispersed in the cytoplasm, had a spherical shape.

Electron microscopy was used to observe the transzonal projections of cumulus cells through the zona pellucida of prepubertal and adult oocytes after follicle collection. The number of corona cell foot projections appeared lower in all prepubertal oocytes compared with adult oocytes (Fig. 1).



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  		FIG. 1. Electron microscopy section of adult (A) and prepubertal (B) oocytes for examination of transzonal projections (pp) of cumulus cells through the zona pellucida.

Protein Labeling and Analysis

Protein labeling was done during the first and last 4 h of maturation in order to analyze differences in the synthesized proteins at the start and end of meiosis. In three replicates we have analyzed 60 and 45 prepubertal and adult oocytes, respectively. Cumulus-complex oocytes derived from prepubertal and adults with equivalent numbers of granulosa cell layers were used in these experiments.

The qualitative pattern of neosynthesized proteins at both 0–4 and 20–24 h of maturation did not show appreciable differences between prepubertal and adult sheep oocytes.

Methionine uptake after labeling (Fig. 2) during 0–4 h of culture was higher (P < 0.01) in cumulus-enclosed oocytes compared with denuded oocytes both in prepubertal (mean ± SD; 7065 ± 1244 vs. 4225 ± 1139 cpm; P < 0.01) and adult (13 092 ± 1211 vs. 2663 ± 432 cpm; P < 0.01) oocytes. In adult oocytes uptake of labeled methionine was significantly higher than in prepubertal ones (13 092 ± 1211 vs. 7065 ± 1244 cpm; P < 0.01) when the cumulus was present, but prepubertal denuded oocytes incorporated more methionine than adult ones (4225 ± 1139 vs. 2663 ± 432 cpm; P < 0.01). Labeled methionine incorporation during 20–24 h of culture was higher in cumulus-enclosed adult oocytes compared with prepubertal (15 558 ± 2258 vs. 9615 ± 676 cpm; P < 0.01) and cumulus-denuded oocytes derived from both adult (15 558 ± 2258 vs. 7529 ± 676 cpm; P < 0.01) and prepubertal (15 558 ± 2258 vs. 9003 ± 1200 cpm; P < 0.01) animals. There were no differences in labeled methionine incorporation between cumulus-enclosed prepubertal oocytes (7529 ± 676 cpm), denuded prepubertal oocytes (9003 ± 1200 cpm), and denuded adult oocytes (9615 ± 676 cpm).



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  		FIG. 2. Methionine uptake in denuded and cumulus-enclosed prepubertal and adult oocytes after labeling during 0–4 and 20–24 h of in vitro culture (mean ± SD).

Biochemical and Biological MPF Activity

Biochemical determination of H1 kinase activity during the meiotic progression (Fig. 3) of prepubertal and adult sheep oocytes (measuring in vitro phosphorylation of H1 kinase activity) showed that the kinetics of MPF activity were similar between prepubertal and adult oocytes. MPF levels were very low at GV stage, rose during GVBD, and chromosome condensation until MI, and dropped to basal levels during the MI-telophase transition. An increase of H1 kinase activity levels occurred as oocytes progressed to MII.



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  		FIG. 3. MPF fluctuations in prepubertal and adult oocytes during in vitro meiotic progression. All bands derive from a single gel. *At 24 h, H1 kinase activity was statistically different, P < 0.001

Prepubertal and adult oocytes displayed similar levels of MPF during meiosis, but at MII stage, the MPF level was significantly lower in prepubertal oocytes (P < 0.001; Fig. 3). In fact, MII prepubertal oocytes showed 80% H1 kinase activity compared with adult oocytes.

Chromatin modification of reporter prophase oocytes after cell fusion was an indicator of biological MPF activity. Reporter GV sheep oocytes were fused with MI and MII stage oocytes derived from prepubertal and adult ovaries. Both MI prepubertal and adult oocytes were able to induce nuclear envelope breakdown and chromosome condensation in GV oocytes 1 h after fusion. The same effect (Fig. 4) was observed after a 1-h culture of fused GV-MII adult oocytes. On the contrary, prepubertal MII oocytes were incapable of inducing GVBD and chromosome condensation 1–2 h after fusion (Table 1).



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  		FIG. 4. Biological activity assay of MPF levels in MII adult (A) and prepubertal (B) oocytes after fusion with sheep GV reporter oocytes. Note the chromosome condensation (PCC) observable in A and the intact GV in B 1 h after fusion


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  		TABLE 1. Fusion of MI and MII prepubertal and adult oocytes with GV reporter oocytes after in vitro culture

DISCUSSION

The results of this work show that oocytes recovered from prepubertal sheep had some significant biological differences compared with adult oocytes. In particular, we found a reduced synthetic activity, probably due to a lower cell coupling between somatic and germinal cell compartments. Moreover, lamb MII oocytes showed significantly lower MPF (P < 0.001) activity tested by H1 kinase assay and by cell fusion experiments. To our knowledge, this represents the first report of MPF fluctuations in prepubertal sheep oocytes.

Reduced synthetic activity could be related to several things: 1) a lower coupling between cumulus cells and the oocyte, as observed after examination of zona pellucida projections by TEM; 2) a less-developed intercellular communication due to a decreasing of the gap junction number; or 3) a reduced volume of lamb oocytes, which as we observed in a previous study [40], had a significantly smaller diameter compared with those derived from ovaries of adult animals.

The association between follicle cells and the oocyte in the form of foot processes was previously studied in lambs by O'Brien et al. [41] who did not observe any differences between prepubertal and adult sheep oocytes. However, they used oocytes derived from 6-mo-old prepubertal animals and at this age, the grade of coupling can be completely defined. In our study, oocytes were derived from very young animals (aged 1 mo) and, consequently, these associations could be not well established.

The decrease in coupling between cumulus cells and oocytes could be attributable to reduced intercellular communication. This hypothesis is supported by our previous experiments which demonstrated that, after injection of Lucifer yellow into lamb and adult oocytes, its passage through the gap junctions to the cumulus cells was reduced in prepubertal oocytes [42].

Follicle size could also affect the grade of the coupling between cumulus cells and oocytes. It has been observed in bovine that the development of transzonal communications is closely related to folliculogenesis increasing during follicular and oocyte growth [9, 43]. Oocytes from prepubertal animals are mainly derived from small-sized follicles. In sheep it has also been observed that follicular size and oocyte dimension can affect the ability of prepubertal and adult oocytes to resume and complete meiosis in vitro [42].

The importance of this bidirectional communication for meiotic resumption and progression has been reported in the hamster, in which the removal of somatic cells at the GV stage determines arrest at MI [44]. Sheep oocytes stripped of cumulus cells showed a significant decrease in biosynthetic activity, actin production in particular, which is one of the major, newly synthesized proteins [45].

A significantly lower incorporation of labeled methionine into proteins is observable in cumulus-enclosed oocytes derived from prepubertal animals compared with those from adults. This can be due to the reduced size of lamb oocytes but also to defective signaling between the cumulus cells and the oocyte. In fact, in experiments in which oocytes were stripped of granulosa cells in the early stages of incubation, the uptake of methionine was equal or slightly higher in prepubertal oocytes than it was in adult oocytes.

In this study we examined for the first time the changes in MPF activity during the maturation of prepubertal sheep oocytes and we compared these levels with those observed in adult oocytes. MPF fluctuations in prepubertal and adult oocytes were similar during meiotic progression, but at MII stage we found that prepubertal oocytes had a lower MPF level compared with that in adult oocytes. This could indicate that during the passage from MI to MII, the machinery that provides for a high MPF is not as efficient as it is in oocytes derived from adult oocytes. A deficiency of cyclin B synthesis can occur at this stage, or defective phosphorylation of the threonine-serine residues could affect the formation of an active form of the MPF complex.

This is confirmed by the fusion experiments in which biological MPF activity was tested. In fact, the results showed that MII oocytes derived from lambs were unable to induce nuclear membrane destabilization and chromosome condensation in the GV partner after 1–2 h post-fusion. The adult oocytes, on the other hand, were fully capable of inducing meiotic resumption in the counterpart after the 1 h post-fusion. The MPF activity is able to complete meiosis but, interestingly, is unable to induce chromosome condensation. A decrease in MPF levels has been reported in aged oocytes and oocytes cultured in suboptimal conditions [46]. However, in prepubertal oocytes, lower MPF activity has been detected just after the telophase transition, and this may indicate that this reduced MPF activity could arise for different reasons. We cannot exclude that synthesis of MPF occurs in prepubertal oocytes at the same levels as in adult oocytes and that molecules that act on the stabilization of the MPF complex are lacking in the prepubertal oocytes. This low stabilization could be responsible for the increase of spontaneous parthenogenetic activation of oocytes from prepubertal subjects [31]. Furthermore, this decline can be related to the decrease in other molecules such as c-mos, which are responsible for the stability of the cytostatic factor, and which could be less functionally active in those oocytes. Authors [47] have shown how oocytes derived from c-mos knockout mice begin to develop without activation stimulus.

In conclusion, oocytes derived from prepubertal ovaries of 4- to 5-wk-old lambs showed some deficiencies compared with those from adults. A decreased presence of transzonal processes and a defective coupling between cumulus cells and oocytes can affect biosynthetic activity. We found that lamb oocytes had the same fluctuations during meiosis as adult oocytes did, but they had a significantly lower level of MPF activity at MII stage. These deficiencies could be responsible for the reduction in the developmental capacity of prepubertal oocytes in sheep.

FOOTNOTES

First decision: 27 November 2000.

1 Supported by the Ministero dell'Università e Ricerca Scientifica e Tecnologica (project ex 40%). Back

2 Correspondence: Sergio Ledda, Department of Animal Biology, University of Sassari, V. Vienna 2, 07100 Sassari, Italy. FAX: 39 79 229429; vetfis{at}ssmain.uniss.it Back

Accepted: February 27, 2001.

Received: October 24, 2000.

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