Directory UMM :Data Elmu:jurnal:A:Animal Reproduction Science:Vol64.Issue1-2.Dec2000:

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Culture of bovine oocytes from small

antral follicles in meiosis-inhibiting medium

with butyrolactone I: RNA synthesis, nucleolar

morphology and meiotic competence

A. Pavlok

∗

_{, J. Kaˇnka, J. Motl´ık, P. Vodiˇcka}

Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Cz-277-21 Libechov, Czech Republic

Received 9 February 2000; received in revised form 3 July 2000; accepted 18 July 2000

Abstract

Bovine oocytes originated from follicles of two different size categories (medium (M), 3–6 mm and small (S), 1–2 mm) were cultured for 24 and 70 h, respectively, in a meiosis-inhibiting medium (MIM) supplemented with 100mM butyrolactone I (BL I). At the end of culture, cumulus oocyte

complexes (COC) from M and S follicles were labeled by3_{H-uridine for 30 min. The}

autoradio-graphy (ARG) of semi-thin sections of COC showed the labeling of the germinal vesicles (GV) as: (1) The COC of the M category labeled immediately after isolation from follicles showed only weak labeling (+) of the GV. The COC of the S category labeled immediately after their isolation showed mostly intensive labeling (++++, +++) of the GV. (2) When the COC were labeled after 24 and 70 h of culture in MIM, no labeling was observed in the M category. The S category of oocytes showed the slightly decreased labeling (+++,++) after 24 h and negligible labeling after 70 h of culture. The pattern of very intensive labeling of granulosa cell nuclei of all mentioned groups was practically not changed during the whole 70 h period of culture in both categories. The nucleolar ultrastructure of S category oocytes revealed time dependent changes from the reticular fibrillogranular structure present in freshly isolated oocytes. The several fibrillar centers before the culture changed to the fibrillogranular appearance with few large and a number of small vacuoles and an exclusively fibrillar area after 24 h of culture. Finally, nucleoli acquired a mostly exclusively fibrillar structure with one large fibrillar center after 70 h of culture. In the second experiment, the meiotic maturation of COC of S category was inhibited in MIM for 48 h. The subsequent 24 h culture in a medium with BOS and gonadotropins resulted in 81.0% oocytes matured to metaphase II (M II). Only 27.1 and 11.3% of the control S oocytes cultured in a medium, with BOS and gonadotropins

∗_{Corresponding author. Tel.:}₊_{42-206-697147; fax:}₊_{42-206-697186.} E-mail address:[email protected] (A. Pavlok).

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directly after isolation, reached M II after 48 or 72 h of culture, respectively. The two-step culture increased significantly the meiotic competence of cattle oocytes isolated from small antral follicles. © 2000 Elsevier Science B.V. All rights reserved.

Keywords:Cattle-reproductive technology; Meiosis; Oocyte; Butyrolactone I; 5-3_{H-uridine; Autoradiography}

1. Introduction

The meiotic competence of the mammalian oocyte develops in close correlation with follicular growth. The formation of the antral cavity seems to be the stage at which the rodent oocytes acquire their full size and concomitant with their meiotic competence (Erickson and Sorensen, 1974). In cattle, the small antral follicles less than 1 mm in diameter possess the growing oocytes usually not exceeding the size of 110mm. Only a limited portion of these oocytes resume meiosis in vitro (for review see Motl´ık, 1989; Fair et al., 1995). The fully grown oocytes (about 120mm in size) with both, meiotic and developmental competence could be obtained from oocytes above 3 mm in size (Pavlok et al., 1992; Fair et al., 1995). The growing bovine oocytes synthesize very intensively ribosomal RNA (rRNA) and the nucleolar morphology is characterized by fibrillogranular elements, fibrillar centers and discrete vacuoles. In oocytes, the gradual acquisition of their full size is accompanied by the distinct compactisation of nucleolar fibrillar material and formation of a single, large fibrillar center (Crozet et al., 1986). These changes are accompanied by termination of rRNA synthesis and reduction of heterogeneous nuclear RNA (hnRNA) synthesis. The above mentioned morphological phenomena are correlated not only with the size of follicles and oocytes but also with the meiotic and developmental competence of oocytes cultured in vitro (Pavlok et al., 1992, 1997).

To simulate more precisely the in vivo maturation pattern of fully grown bovine oocytes, a two-step in vitro culture protocol was tested. In the first step, the oocytes were cultured in a meiosis-inhibiting medium (Lonergan et al., 1997; Avery and Greve, 1997; Saeki et al., 1997; Motl´ık et al., 1998) to prolong the time of the transcriptional and translational activity specified for the GV stage (Kastrop et al., 1990; Pavlok et al., 1997). Hyttel et al. (1997) proposed for this first period of culture under the technical term “capacitation”. After this meiosis-inhibiting period, the oocytes are transferred for 24 h to the classical maturation medium.

A special prolonged culture protocol for in vitro growth and subsequent maturation of oocytes from preantral follicles was first used in a mouse (Eppig and Schroeder, 1989). The subsequent fertilization, embryo culture and their transfer resulted in some newborn mice. Quite recently, Yamamoto et al. (1999) cultured bovine oocytes isolated from early antral follicles by the modified method of Harada et al. (1997) and obtained a new born calf after their fertilization and embryo transfer to a recipient. A new methodological approach to the culture of ovine early antral follicles was developed by Newton et al. (1999). However, the proportion of successfully maturing oocytes was still very low.

In this study, RNA synthesis of bovine meiotically competent and incompetent oocytes was analyzed before and after their culture in a meiosis-inhibiting medium with butyro-lactone I (BL I) as already described by Motl´ık et al. (1998) and Kubelka et al. (2000).

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Concomitantly, ultrastructural changes of nucleoli from meiotically incompetent oocytes were followed. To increase the meiotic competence of oocytes from 1 to 2 mm follicles (S category), the two-step culture, in the meiosis-inhibiting medium (with BL I) and subse-quently in the meiosis-stimulating medium was tested.

2. Material and methods

2.1. Animals, follicles, oocytes, culture

Bovine ovaries, obtained at a local slaughterhouse, were rinsed and collected in physio-logical saline at 25–30◦_{C. The ovaries were transported to the laboratory within 1 h, rinsed}

briefly in ethyl alcohol and washed twice in saline. The follicles were dissected and separated into two categories, medium (M) 3–6 mm in diameter and small (S) 1–2 mm in diameter, twice washed in saline and placed into 9 mm Petri dishes with a culture basic medium (BM) of the following composition: TCM 199 (Sevac, Praha, Czech Republic)×10 conc., 9.4 ml; NaHCO₃ 7.5% solution (Sevac), 2.1 ml; HEPES acid, 9.5 mM; Na-pyruvate, 1.82 mM; polyvinyl alcohol (PVA), 3 mg/ml−1_{; penicillin K-salt, 50 IU/ml}−1_{; streptomycin sulfate,}

50 IU/ml−1_{; amphotericin B, 125 ng/ml}−1_{(all from Sigma) supplemented with deionized}

and nanopure filtered H2O to 100 ml. The oocytes were isolated from the follicles with

fine forceps and preparation needle, picked up and transferred to the 500ml of BM supple-mented with butyrolactone I (Funakoshi, Tokyo, Japan), 100mM, called meiosis-inhibiting medium (MIM) and cultured in 4-well Nunclon dishes (Roskilde, Denmark) under min-eral oil (Serva, Heidelberg, Germany) for either for 24 or 70 h. For the second experi-ment the same MIM was suppleexperi-mented with BSA (Serva), 3 mg/ml−1_{. For the second step}

of the culture, the COC were washed four times in BL I free BM and cultured 24 h in meiosis-stimulating medium with gonadotropins (MSM) (Pavlok et al., 1997). The control group of COC of S category was cultured directly after isolation in MSM for 48 and 72 h, respectively. All cultures were carried out at 39◦_{C, in atmosphere of 5% CO}

2, 10% O2, and

85% N₂.

Instat software was used for statistical evaluation (GraphPad Software). The effect of BL I on meiotic maturation was evaluated byχ2analysis with Yates (continuity) correction.

2.2. Labeling of oocytes, autoradiography and electron microscopy

Immediately after COC isolation or after the appropriate time of their culture, the oocytes were transferred to the same culture medium enriched with 5-3H-uridine (Amersham, Little Chalfont, England, spec activity 40 MBq/mM−1_{), 7.4 MBq/ml}−1_{for 30 min. At the end of}

labeling, the COC were washed three times in nonradioactive medium at 4◦_{C and fixed in}

0.6% paraformaldehyde(Sigma)+2.5% glutaraldehyde (Sigma), mixture with cacodylate

(Sigma), buffer (pH 7.4). After washing in this buffer at 4◦_{C the oocytes were post-fixed for}

60 min in 2% OsO₄(Sigma) dehydrated in ethanol and embeded in Polybed (Polysciences, Warrington, FL, USA). From individual oocytes, semi-thin sections (1mm) were prepared for autoradiography and thin sections for electron microscopy.

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Semi-thin sections were coated with nuclear liquid emulsion Ilford K5 (Mobberley, Eng-land) and exposed at 4◦_{C for 30 days. After development with Kodak solution D 19b}

(Rochester, NY, USA) at 19◦_{C for 4 min and fixation (Baserga and Malamud, 1969) the}

autoradiograms were stained with methylene blue before examination.

The labeling intensity was examined by light microscopy. The images were captured with SenSys CCD camera (Photometrics Ltd., Tuscon, AZ) on Olympus AX70 microscope (Olympus Optical Co. Ltd., Tokyo, Japan) using 100×objective. Ratio of grain to nuclear area expressed in percent units was measured using Olympus Micro Image software. The sorting of oocytes according to the proportion of GV cowered with silver grains was as follows. The GV with 65–80% of grain area were evaluated as++++, 45–64% as+++, 15–44% as++, 3–14% as+. About 1–2% represented the background and was evaluated as below.

For electron microscopy, a few thin sections practically of all oocytes used for autora-diographic analysis were prepared on grids, contrasted with uranyl acetate for 30 min and with lead citrate for 10 min and examined under JEOL 1 200 EX electron microscope at 80 kV. The most representative photos of S category are presented in the manuscript.

3. Results

3.1. RNA synthetic activity of S and M oocytes

As seen from the Table 1, the oocytes from COC of the M category labeled with3_H-uridine

immediately after isolation showed only weak labeling (+) of the GV area. After 24 and 70 h of culture in the meiosis-inhibiting medium, the oocytes of the same category showed no labeling of the GV area. However, the nuclei of the granulosa cells were intensively labeled in all groups. The oocytes of the S category labeled immediately after isolation (before culture) showed intensive labeling (++++,+++) of GV area (Fig. 1). When the COC of the same category were labeled after 24 h of culture, a slightly decreased tendency of labeling of GV area (mostly+++,++) was observed (Figs. 2 and 3). After 70 h of culture, the GV area was labeled only slightly above the background (Fig. 4). Also the COC

Table 1

Labeling of nuclei of bovine oocytes with 5-3_{H-uridine before and after 24 and 70 h culture in a meiosis-inhibiting} medium with butyrolactone Ia

Oocyte category Time of culture (h) Labeling intensity and no. of oocytes Total no. of oocytes

++++ +++ ++ + 0

M (3–6 mm) 0 0 0 0 2 1 3

24 0 0 0 0 4 4

70 0 0 0 0 3 3

S (1–2 mm) 0 4 3 1 0 0 8

24 0 2 2 1 1 6

70 0 0 0 1 2 3

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Fig. 1. Light microscopy radiography of oocytes from two size categories of follicles (M, 3–6 mm and S, 1–2 mm in diameter). Semi-thin sections of oocytes labeled for 30 min with 5-3_{H-uridine after 0, 24 and 70 h of culture} in meiosis-inhibiting medium supplemented with 100mM BL I. Thirty days,exposition under the nuclear liquid

emulsion (×600). Heavily labeled (dark) GV area of oocyte and surrounding granulosa cells. S category oocyte

before culture (labeling intensity evaluated as++++).

Fig. 2. Light microscopy radiography of oocytes from two size categories of follicles (M, 3–6 mm and S, 1–2 mm in diameter). Semi-thin sections of oocytes labeled for 30 min with 5-3_{H-uridine after 0, 24 and 70 h of culture} in meiosis-inhibiting medium supplemented with 100mM BL I. Thirty days,exposition under the nuclear liquid

emulsion (×600). Heavily labeled GV area mainly around nucleoli of 24 h cultured S category oocyte (labeling

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Fig. 3. Light microscopy radiography of oocytes from two size categories of follicles (M, 3–6 mm and S, 1–2 mm in diameter). Semi-thin sections of oocytes labeled for 30 min with 5-3_{H-uridine after 0, 24 and 70 h of culture} in meiosis-inhibiting medium supplemented with 100mM BL I. Thirty days,exposition under the nuclear liquid

emulsion (×600). Mild labeling of GV area of 24 h cultured S category oocyte (labeling intensity evaluated as ++). The granulosa cells are heavily labeled.

Fig. 4. Light microscopy radiography of oocytes from two size categories of follicles (M, 3–6 mm and S, 1–2 mm in diameter). Semi-thin sections of oocytes labeled for 30 min with 5-3_{H-uridine after 0, 24 and 70 h of culture} in meiosis-inhibiting medium supplemented with 100mM BL I. Thirty days,exposition under the nuclear liquid

emulsion (×600). Only significantly more grains than the background of GV area of 70 h cultured S category

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Fig. 5. Fine structure of nucleoli in oocytes of S category before culture, after 24 and 70 h culture in meiosis-inhibiting medium (MIM) with 100mM of BL I as seen in Material and methods. Nucleolus from the S

category oocyte of reticular structure with several fibrillar centers (arrows) fixed immediately after isolation from the follicle (×2500).

of S category showed the intensive labeling of granulosa cells nuclei during the whole 70 h of culture.

3.2. Nucleolar ultrastructure of S category oocytes: freshly isolated, 24 and 70 h cultured with BL I

Immediately after isolation of S category oocytes, the nucleoli possessed a reticular fibrillogranular structure with numerous fibrillar centers (arrows) (Fig. 5). After 24 h culture in MIM, fibrillogranular structure was shifted peripherally by several large vacuoles. A number of small vacuoles and exclusively fibrillar area (arrow) were observed (Fig. 6). After 70 h of culture in MIM, nucleoli were exclusively fibrillar with one large fibrillar center (arrow) (Fig. 7).

3.3. Two-step culture of S category oocytes

Comparing with the one-step maturation of S category oocytes, the two-step culture of these oocytes (in MIM and subsequently in MSM have the capability to increase signif-icantly (P < 0.01) their meiotic competence (Table 2). While after the one-step 48 and

72 h maturation of control COC in MSM, only 27.1 and 11.3% matured to M II stage, respectively, two-step culture of experimental COC resulted in 81.0% maturing to M II. In the control group, of COC matured 48 and 72 h, 67.8 and 54.7% did not proceed beyond the metaphase I (M I)–anaphase I block stage, respectively. However, in both the control and experimental groups, some proportions of oocytes (5.1, 30.2 and 7.9%, respectively) remained either in the GV or late diakinesis (LD) stages.

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Fig. 6. Fine structure of nucleoli in oocytes of S category before culture, after 24 and 70 h culture in meiosis-inhibiting medium (MIM) with 100mM of BL I as seen in Material and methods. Nucleolus from the S

category oocyte fixed 24 h after culture in MIM. The fibrinogranullar structure is accompanied with some large vacuoles, more small vacuoles and one fibrillar area (arrow) (×3500).

Fig. 7. Fine structure of nucleoli in oocytes of S category before culture, after 24 and 70 h culture in meiosis-inhibiting medium (MIM) with 100mM of BL I as seen in Material and methods. Nucleolus of the S

category oocyte fixed 70 h of culture in MIM. Compact fibrillar material is accompanied with a large single fibrillar center (arrow) (×8000).

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Table 2

Meiotic competence of bovine oocytes from a small category of oocytes (1–2 mm in follicle diameter) cultured directly 48 h in meiosis-stimulating medium (MSM) with gonadotropins (control group-C1), or in two-step pro-tocol, in the first step 48 h in MSM without gonadotropins and in the second step in MSM with gonadotropins (C2)a

Group Time of culture (h) in Total no. of COC

GV–LDb_{(%) M I anaphase I} block (%)

Activated pronuclei (%)

M II (%) MIM MSM

C1 – 48 59 3 (5.1) a 40 (67.8) a 0 16 (27.1) a

C2 – 72 53 16 (30.2) b 29 (54.7) a 2 (3.8) 6 (11.3) a

Exp. 48 24 63 5 (7.9) a 7 (11.1) b 0 51 (81.0) b

a_{The experimental group (Exp.) was cultured in the first step 48 h in meiosis-inhibiting medium (MIM) and} in the second step 24 h in MSM with gonadotropins. The data in the same column with different letters differ significantly (P <0.05).

b_{Late diakinesis.}

4. Discussion

The intensity of maternal RNA synthesis in oocytes correlates well with the stage of their growth and with the corresponding changes in morphology of nucleoli (Motl´ık et al., 1984b; Crozet et al., 1986). The RNA synthetic activity decreases gradually as the oocytes reach their full size. These morphological and functional modifications seem to be the most important processes for acquisition of their full meiotic competence (Motl´ık et al., 1984a). In this connection, it is quite interesting that the nucleoli of cattle oocytes from the S category cultured for 70 h resembled nucleoli of the M category (see Crozet et al., 1986). The philosophy of meiosis inhibition during in vitro culture is to prolong the maternal RNA synthesis and a protein synthesis which is specific for GV stage oocytes (Kastrop et al., 1990; Pavlok et al., 1997). The importance of this stage for the improvement of the developmental competence of growing bovine oocytes was documented by Blondin et al. (1997). Using several germinal vesicle breakdown (GVBD) inhibitors for the first step of culture, the developmental competence of fully grown bovine oocytes did not exceed the results obtained with one-step maturation protocol (Lonergan et al., 1997; Avery and Greve, 1997; Saeki et al., 1997; Mermillod et al., 2000).

Several experiments on somatic and malignant cells proved that BL I is a selective in-hibitor of cyclin dependent kinases. It inhibits both cdk2 and cdc2 kinases and blocks the cell cycle at the G1/S and G2/M transitions (Kitagawa et al., 1994, 1995; Nishio et al., 1996). The kinetic studies of BL I showed that it inhibits cdc2 kinase by competing with ATP, not with the substrate (Kitagawa et al., 1993).

Recently, it has been demonstrated (Motl´ık et al., 1998) that BL I can effectively inhibit GVBD of fully grown oocytes in several mammalian species. However, a recent article of Hida et al. (1999) reported a dose dependent inhibitory effect of BL I on the synthesis of mRNAs for albumin and alpha-fetoprotein in human hepatoma cells. But the present paper is the first report where the possible effect of BL I treatment on the RNA synthesis of growing bovine oocytes was analyzed. At present, it is difficult to decide if BL I has any detrimental effect on RNA synthesis in growing oocytes. The presented results show a slightly decreasing tendency, in RNA synthesis after 24 h of culture with BL I, and rather

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strongly decreasing after 70 h culture. This decreasing tendency in RNA synthetic activ-ity correlates well with the changes of the nucleolar ultrastructure as also observed in in vivo growing oocytes by (Crozet et al., 1986; Fair et al., 1995). Whether the described morphological changes are a normal prerequisite accompanying full meiotic competence acquisition or rather degenerative ones, as the consequence of long lasting and inconve-nient culture protocol, is partly answered by the results of two-step culture of S category COC (Table 2). In contrast to one-step culture protocol of control COC and to previous experiments (Pavlok et al., 1997), the two-step culture improved significantly the meiotic competence of growing oocytes. The two-step culture of fully grown oocytes also at least partially improved their meiotic and fertilization competence (Kubelka et al., 2000). Quite recently, the first experiments with fertilization of oocytes from follicles 2 mm and less in diameter show some improvement in fertilization competence of experimental group comparing with the control oocytes (Pavlok et al., unpublished).

In conclusion, the results presented in this paper suggest that bovine growing oocytes cultured in the BL I supplemented medium continue in rRNA and hnRNA synthesis. The gradual transformation of fibrillogranular nucleolus to the compact one was accompanied with the decrease of RNA synthetic activity. The second culture step in MSM proved that the full inhibition of meiosis in the BL I supplemented medium during the first culture step enabled cattle growing oocytes to acquire meiotic competence. Further experiments testing penetration and developmental competence are in progress.

Acknowledgements

The authors wish to thank V. Pech, J. Zelenková and Š. Hladký for their technical help in this research. This work was supported by Grant No. 594/96/K162 of Grants Agency of the Czech Republic, EU Grant No. QLRT-1999-00114 and Grant EP 7159 of the Czech Ministry of Agriculture.

References

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competence is acquired before maturation. Theriogenology 47, 1061–1075.

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Graaffian follicles. J. Exp. Zool. 190, 123–127.

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Hida, D., Nakata, K., Shima, Y., Nakao, K., Kato, Y., Ishii, N., Eguchi, K., 1999. Suppression of albumin and alpha-fetoprotein gene expression by butyrolactone I, a selective inhibitor of cdk family, in HuH-7 human hepatoma cells. Anticancer Res. 18, 4317–4322.

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emulsion (×600). Mild labeling of GV area of 24 h cultured S category oocyte (labeling intensity evaluated as ++). The granulosa cells are heavily labeled.

emulsion (×600). Only significantly more grains than the background of GV area of 70 h cultured S category oocyte (labeling intensity evaluated as+). Most of the granulosa cells are heavily labeled.

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category oocyte of reticular structure with several fibrillar centers (arrows) fixed immediately after isolation from the follicle (×2500).

of S category showed the intensive labeling of granulosa cells nuclei during the whole 70 h

of culture.

3.2. Nucleolar ultrastructure of S category oocytes: freshly isolated, 24 and 70 h cultured

with BL I

Immediately after isolation of S category oocytes, the nucleoli possessed a reticular

fibrillogranular structure with numerous fibrillar centers (arrows) (Fig. 5). After 24 h culture

in MIM, fibrillogranular structure was shifted peripherally by several large vacuoles. A

number of small vacuoles and exclusively fibrillar area (arrow) were observed (Fig. 6).

After 70 h of culture in MIM, nucleoli were exclusively fibrillar with one large fibrillar

center (arrow) (Fig. 7).

3.3. Two-step culture of S category oocytes

Comparing with the one-step maturation of S category oocytes, the two-step culture of

these oocytes (in MIM and subsequently in MSM have the capability to increase

signif-icantly (

P <

0 .

01) their meiotic competence (Table 2). While after the one-step 48 and

72 h maturation of control COC in MSM, only 27.1 and 11.3% matured to M II stage,

respectively, two-step culture of experimental COC resulted in 81.0% maturing to M II. In

the control group, of COC matured 48 and 72 h, 67.8 and 54.7% did not proceed beyond

the metaphase I (M I)–anaphase I block stage, respectively. However, in both the control

and experimental groups, some proportions of oocytes (5.1, 30.2 and 7.9%, respectively)

remained either in the GV or late diakinesis (LD) stages.

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category oocyte fixed 24 h after culture in MIM. The fibrinogranullar structure is accompanied with some large vacuoles, more small vacuoles and one fibrillar area (arrow) (×3500).

category oocyte fixed 70 h of culture in MIM. Compact fibrillar material is accompanied with a large single fibrillar center (arrow) (×8000).

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Table 2

Group Time of culture (h) in Total no. of COC

GV–LDb_{(%) M I anaphase I} block (%)

Activated pronuclei (%)

M II (%) MIM MSM

C1 – 48 59 3 (5.1) a 40 (67.8) a 0 16 (27.1) a

C2 – 72 53 16 (30.2) b 29 (54.7) a 2 (3.8) 6 (11.3) a

Exp. 48 24 63 5 (7.9) a 7 (11.1) b 0 51 (81.0) b

b_{Late diakinesis.}

4. Discussion

The intensity of maternal RNA synthesis in oocytes correlates well with the stage of

their growth and with the corresponding changes in morphology of nucleoli (Motl´ık et al.,

1984b; Crozet et al., 1986). The RNA synthetic activity decreases gradually as the oocytes

reach their full size. These morphological and functional modifications seem to be the

most important processes for acquisition of their full meiotic competence (Motl´ık et al.,

1984a). In this connection, it is quite interesting that the nucleoli of cattle oocytes from the

S category cultured for 70 h resembled nucleoli of the M category (see Crozet et al., 1986).

The philosophy of meiosis inhibition during in vitro culture is to prolong the maternal

RNA synthesis and a protein synthesis which is specific for GV stage oocytes (Kastrop

et al., 1990; Pavlok et al., 1997). The importance of this stage for the improvement of the

developmental competence of growing bovine oocytes was documented by Blondin et al.

(1997). Using several germinal vesicle breakdown (GVBD) inhibitors for the first step of

culture, the developmental competence of fully grown bovine oocytes did not exceed the

results obtained with one-step maturation protocol (Lonergan et al., 1997; Avery and Greve,

1997; Saeki et al., 1997; Mermillod et al., 2000).

Several experiments on somatic and malignant cells proved that BL I is a selective

in-hibitor of cyclin dependent kinases. It inhibits both cdk2 and cdc2 kinases and blocks the

cell cycle at the G1/S and G2/M transitions (Kitagawa et al., 1994, 1995; Nishio et al.,

1996). The kinetic studies of BL I showed that it inhibits cdc2 kinase by competing with

ATP, not with the substrate (Kitagawa et al., 1993).

Recently, it has been demonstrated (Motl´ık et al., 1998) that BL I can effectively inhibit

GVBD of fully grown oocytes in several mammalian species. However, a recent article

of Hida et al. (1999) reported a dose dependent inhibitory effect of BL I on the synthesis

of mRNAs for albumin and alpha-fetoprotein in human hepatoma cells. But the present

paper is the first report where the possible effect of BL I treatment on the RNA synthesis

of growing bovine oocytes was analyzed. At present, it is difficult to decide if BL I has

any detrimental effect on RNA synthesis in growing oocytes. The presented results show a

slightly decreasing tendency, in RNA synthesis after 24 h of culture with BL I, and rather

(5)

COC (Table 2). In contrast to one-step culture protocol of control COC and to previous

experiments (Pavlok et al., 1997), the two-step culture improved significantly the meiotic

competence of growing oocytes. The two-step culture of fully grown oocytes also at least

partially improved their meiotic and fertilization competence (Kubelka et al., 2000). Quite

recently, the first experiments with fertilization of oocytes from follicles 2 mm and less

in diameter show some improvement in fertilization competence of experimental group

comparing with the control oocytes (Pavlok et al., unpublished).

In conclusion, the results presented in this paper suggest that bovine growing oocytes

cultured in the BL I supplemented medium continue in rRNA and hnRNA synthesis. The

gradual transformation of fibrillogranular nucleolus to the compact one was accompanied

with the decrease of RNA synthetic activity. The second culture step in MSM proved that

the full inhibition of meiosis in the BL I supplemented medium during the first culture step

enabled cattle growing oocytes to acquire meiotic competence. Further experiments testing

penetration and developmental competence are in progress.

Acknowledgements

The authors wish to thank V. Pech, J. Zelenková and Š. Hladký for their technical help

in this research. This work was supported by Grant No. 594/96/K162 of Grants Agency of

the Czech Republic, EU Grant No. QLRT-1999-00114 and Grant EP 7159 of the Czech

Ministry of Agriculture.

References