Bovine oocytes from early antral follicles grow to meiotic competence in vitro: Effect of FSH and hypoxanthine

ELSEVIER

BOVINE GOCYTES FROM EARLY ANTRAL FOLLICLES GROW TO MEIOTIC COMPETENCE IN VITRO: EFFECT OF FSH AND HYPOXANTHINE M. Harada,l T. Miyano,aa K. Matsumura,l S. Osaki,l M. Miyakel and S. Kato2 1The Graduate School of Science and Technology &&oratory of Animal Breeding and Reproduction Faculty of Agriculture, Kobe University Nada-ku, Kobe 657, Japan Received for publication: September 24, Accepted: June 3, 1997

1996

ABSTRACT A large number of oocytes are contained in the mammalian ovary. A very small number of these oocytes grow to the final size, mature, and are ovulated. In the ovary there are more eady antral follicles than late antral or preovulatory follicles, offering a large pool of oocytes for IVM and IVF if appropriate culture conditions could be devised. In the present study, early antral follicles containing oocytes 90 to 99 pm in diameter were isolated from bovine ovaries. Cumulus-oocyte complexes (COC) with pieces of parietal granulosa (COCG) were then dissected from the follicles. The COCGs were embedded in collagen gels and cultured in Medium 199 with 10% fetal calf serum (FCS) for 8 d. In Experiment 1, the effect of hypoxanthine and FSH on the growth of bovine oocytes was examined. When hypoxanthine (2 and 4 mM) and FSH (10 ng/ml) were added to the culture medium, the number of granulosa cell-enclosed oocytes increased significantly (P&OS). All of the oocy-tes surrounded by granulosa cells showed a normal morphology and were at the germinal vesicle stage, while 75 to 94% of the denuded oocytes were degenerated and had resumed meiosis. The mean diameter of the oocytes showing normal morphology was significantly higher than that measured before culture (P-zz0.05).In Experiment 2, the maturational competence of in vitro-grown bovine oocytes was examined. Oocytes which were 90 to 99 pm in diameter before culture did not have meiotic competence. After being in a growth culture of 4 mM hypoxanthine- and 10 ng/ml FSH-supplemented medium for 7 or 11 d, gtanulosa cell-enclosed oocytes were recovered from the COCGs. No significant difference (P&.05) in the diameters of the oocytes was observed between 7 and I1 d of culture (7 d: 107.5 f 6. I pm, n=30; I1 d: 108.0 f 5.3 pm, n=35). After a subsequent 24 h in a maturation free of hypoxanthine and FSH medium, only 17% of the oocytes cultured for 7 d underwent germinal vesicle breakdown. On the other hand, 89% of the oocytes cultured for 11 d underwent germinal vesicle breakdown, and 11% of the oocytes emitted the first polar body and reached metaphase II. These results demonstrate for the first time that bovine oocytes harvested from early antral follicles can grow, and acquire meiotic competence in vitro. 0 1997 by Elsevier Science Inc

Key words: bovine, early antral follicle, oocyte growth in vitro, hypoxanthine, FSH Acknowledgments The authors thank Drs. John J. Eppig, Yuji tfirao and Radek Proch&ka for helpful suggestions, and the staff of Kobe Meat Inspection Office and Kakogawa ET Center for supplying bovine ovaries. This work was supported in part by grants for scientific research (No. 08660344 and 08556046) from the Ministry of Education, Science and Culture of Japan. a Correspondence and reprint requests. Thenogenology 48:743-755, 1997 0 1997 by Elsevier Science Inc.

0093691Xf97/$17.00 PII SOO93-691X(97)00298-7

Theriogenology

744

INTRODUCIION A large number of small oocytes are contained in mammalian ovaries (17); In the cow the number is estimated to be approximately 120,000 (12). In the primordial follicles, a small population of non-growing oocytes, which are approximately 30 ,um in diameter, begins to grow and reach a final size of 120 pm (13). During the growth of the oocyte, the surrounding granulosa cells proliferate and the follicle increases in size (19). In turn, some fully-grown oocytcs are selected to resume meiosis and reach metaphase II. Ovulation then occurs under the influence of gonadotropins. A large number of remaining oocytes do not enter the growth phase or degenerate in the ovary. Eppig (5) has reported that developing mouse oocytcs can grow to their final size in culture when the oocytes are able to maintain junctional communication with surrounding granulosa cells. He and his colleague have successfully produced living young from in vitro grown, matured and fertilized mouse oocytcs (10). Recently, Eppig and 0’3rien demonstrated that mouse oocytes in primordial follicles grew to the final size, underwent fertilization in vitro, and developed into a liveborn pup (9). Their results show that the complete development of mouse oocytes from the primordial to the mature stage is possible in vitro. For livestock production, the culture of small oocytes could provide a large population of female germ cells. However, culture systems for growing oocytes from larger species have not yet been established. Hirao et al. (18) have reported that pig oocytes harvested from preantral follicles in the mid-growth stage (70 to 90 pm) grew to their final size (120 pm) and matured to metaphase II in vitro. In the bovine, it has been reported that a proliferation of granulosa cells from preantral follicles can be stimulated by administration of basic FGF, FSH and EGF, and that the follicles survive and are steroidogenically active after 6 d in culture (28). Fegueiredo et al. have reported that the mean diameter of bovine preantral follicles increases after 5 d in culture (14). However, the growth of bovine oocytes was not the focus of these reports. Bovine oocytes grow to a volume 3.54 times larger than those of mouse oocytes, and they continue to grow in follicles that have formed an antrum. The smallest bovine antral follicles (0.13 mm in diameter) containing growing oocytes require about 42 d to develop to their preovulatory size (21). On the other hand, mouse oocytes in primordial follicles take only 2 wk to grow to their final size (16). Considering these differences, different culture conditions may be required for the growth of bovine oocytes in vitro. Hypoxanthine, a naturally occurring CAMP-phosphodiesterase inhibitor, is a component of pig and mouse follicular fluid (4, 11). It maintains the association between the mouse oocyte and surrounding granulosa cells in vitro (7). It has been reported that the number of morphologically normal oocytes increases when bovine preantraI follicles are cultured in hypoxanthinesupplemented medium (14). FSH has reported to promote follicle development in cultured bovine preantrai follicles (20,24,28). In this study, we cultured bovine oocytes from early antral follicles in hypoxanthine- and FSH-supplemented medium, and examined the growth of the oocytes and the meiotic competence to mature to metaphase II. MATERIALS

AND METHODS

Collection of Bovine Early Antral Follicles Ovaries were obtained from pure-bred and cross-bred Japanese Black cows slaughtered at a local abattoir. Following 3 washes in Dulbecco’s phosphate buffered saline, early and follicles were dissected from the ovarian cortex while immersed in TCM199b (pH 7.4) containing 0.1% polyvinyialcohol, 0.85 mg/ml NaHC03,0.08 mg/ml kanamycinc, and 25 mM HEPES. Because an

Theriogenology

745

antrum was first observed under a dissection microscope in follicles larger than 0.4 mm in diameter, folIicles of 0.5 to 0.7 mm in diameter were defined as early antral follicles for this study, Connective tissue surrounding the follicles was stripped off using forceps, and only early antral follicles, in which no granulosa cells were detached from the follicle walls, were selected with a dissection microscope (x 40). We routinely dissected 30-40 follicles from 10 ovaries for each experiment. About one third of the collected follicles contained degenerated granulosa cells. some ovaries containing IO while others containing only a few. Cumulus-oocyte complexes with pieces of parietal granulosa (COCGs) were dissected from the selected follicles using fine forceps and a 25-G needle. During the collection of the COCGs, some cumulus-oocyte complexes were detached from the parietal granulosa. We used only intact COCGs for our study. Each COCG was transferred into a IO-pi drop of HEPES-buffered TCM I99 under paraffin oil, and the diameter of each oocyte (excluding zona pellucida) in the COCGs was measured to the nearest I pm with an ocular micrometer attached to an inverted phase contrast microscope. Growth Culture of COCGs Culture of bovine COCGs was based on the method described by Hirao et al. (18). After measurement of the oocyte diameter, COCGs containing oocytes of 90 to 99 pm in diameter were selected and pooled in HEPES-buffered TCM199. The COCGs were divided randomly into experimental groups and embedded in collagen gels following 2 washes in HEPES-buffered TCM199. A collagen mixture was made by mixing a 0.3% acid collagen solution,d IO times concentrated TCM199, and 0.05 N sodium hydroxide solution containing 22 mg/ml NaHCO3 and 47.7 mg/ml HEPES, at a ratio of 8:l:l (v:v:v). A 03ml aliquot of the mixture was placed in a Petri dish, and 2 to 6 of the COCGs were put into the mixture with a small volume of medium. Another 0.3 ml of the collagen mixture was then poured over the COCGs. The gels including the COCGs were then placed into an incubator at 39 “C for 20 min. After gelatinization, 4 ml of culture medium was poured onto the gels containing the COCGs, which were then cultured at 39 “C in a humidified atmosphere of 5% CO2 and 95% air. The basic culture medium was TCM199 containing 10% FCS,f 2.2 mg/ml NaHCQ, 0.08 mg/ml kanamycin and 0.1 mgiml sodium pyruvate. In Experiment 1, either 2 or 4 mM hypoxanthineg were added to the basic culture medium; while in Experiment 2, either IO or 100 ng/ml FSHh were added to the 4 mM hypoxanthine-supplemented culture medium. The COCGs were cultured for 8 d, and half of the culture medium volume was changed to fresh medium after 4 d. After the growth cuiture, the collagen gels containing COCGs were digested with 0.1% collagenasei for 15 min, and COCGs were recovered. Because some COCGs formed an antral follicle-like structure, the oocytes enclosed by granulosa cells were collected from these structures using forceps and a fine needle and completely denuded by pipetting. They were then transferred into IO-,ul drops of HEPES-buffered TCM199. Oocytes which showed evidence of cytoplasmic or other forms of degeneration were excluded from further analysis. Oocytes showing normal morphology were considered surviving oocytes. After measurement of the diameters, the oocytes were mounted onto slides, fixed in acetic ethanol (1:3), stained with 1% aceto-orcein. and examined under a differential interference microscope at x 400 magnification. b Nissui Pharmaceutical Co. Ltd., Tokyo, Japan. c Sigma, St Louis, MO, USA. d Cellmatix Type I, Nitta Gelatine, Osaka, Japan. e #1008, Falcon, NJ, USA. f Bio Cell Co. Ltd., CA, USA. g Kohjin Co. Ltd., Tokyo, Japan. h from porcine pituitary, UCB-Bioproducts, Belgium. 1from Clostridium histolyticum, Wako Pure Chemical Industries, Ltd., Osaka, Japan.

746

Maturation Culture of In Vitro Grown Oocytes Cumulus-oocyte complexes with pieces of parietal granulosa collected from early autral follicles were embedded in collagen gels and cultured for 7 or 11 d as described above. The culture medium was the basic culture medium containing 4 mM hypoxanthine and 10 @ml FSH. Every fourth day, half of the culture medium volume was replaced by fresh medium. After the growth culture, only granulosa cell-enclosed oocytes were recovered and the diameters of the oocytes were measured as described above. Some of the granulosa cell-enclosed oocytes cultured for 11 d were denuded by pipetting, and then fixed and stained, after which the nuclear morphology was examined. The granulosa cell-enclosed oocytes were washed in the basic medium and were further cultured individually in a l@,ul drop of the medium for 24 h at 39 ‘C in a humidified atmosphere of 5% CO2 and 95% air. Granulosa cell-enclosed oocytes isolated from ovarian antral follicles of 0.5 to 0.7 mm in diameter were used as a control. After measurement of oocyte diameters, the granulosa cell-enclosed oocytes in the control group were cultured in the same manner for 24 h. After the maturation culture, the oocytes were denuded, stained, and examined as described above. The precise maturational stage of each oocyte was determined based on the changes in configuration of chromosomes and nuclear membranes (23). Oocytes with abnormal configurations of condensed chromosomes were classified as degenerated oocytes. Statistical Analyses For assessing the effects of hypoxanthine or FSH on the growth of bovine oocytes, 6 independent experiments were conducted. For assessing the maturational competence of in vitrogrown oocytes, 4 independent experiments were conducted. Results from all replicates were pooled and analyzed. Statistical differences in the mean diameters of the oocytes were analyzed by the Student’s t-test. Other values were analyzed by a chi-square test. A P value 4.05 was considered to be statistically significant. RESULTS Effect of Hypoxanthine on Growth of Bovine Oocytes After 1 d of culture, COCG parietal gtanulosa cells enclosed the cumulus-oocyte complexes, and the COCGs changed to a spherical shape in all groups. In the hypoxanthine-free medium, 60 to 70% of the COCGs formed au antrum-like structure after 3 to 4 d of culture; thereafter, the granulosa cells began to spread into collagen gel (Figure la). After 8 d of culture, all COCGs spread into the gel and no antrum-like structures remained in this group. In the hypoxanthinesupplemented medium, 60 to 70% of the COCGs also formed at&rum-like structures after 3 to 4 d of incubation, but the structures were maintained after 8 d of culture in 28% (8/29) and 45% (15133) of the COCGs exposed to 2 and 4 mM hypoxanthine, respectively (Figure lb). After 8 d of culture, the collagen gels were digested and COCGs were recovered. Of the cultured COCGs, 42.48 and 70% of the oocytes were morphologically normal, and had increased mean diameters from 90-99 pm to 107.1 f 8.3, 109.1 f 9.0 and 111.7 f 9.7 pm in media containing 0,2 and 4 mM of hypoxanthine, respectively (Table 1). Each of the mean diameters was significantly higher (PcO.05) than those measured before culture. All of the oocytes recovered from the antrum-like structures were enclosed by granulosa cells and showed no signs of degeneration (Table 2). Of the COCGs which did not maintain their antrum-like structure, most of the oocytes were denuded. Only one granulosa cell-enclosed oocyte was recovered from the COCGs that did not maintain their antrum-like structure after culture. Approximately half of the denuded oocytes showed signs of degeneration. All of the granulosa cell-enclosed oocytes were at the germinal vesicle (GV) stage. In contrast, some of the denuded oocytes had resumed meiosis; and 38% (5/13), 67% (4/6) and 88% (7/8) had undergone germinal vesicle breakdown (GVBD) in the media supplemented with 0,2 and 4 mM hypoxanthine, respectively.

Theriogenology

747

Figure 1. Cultured bovine cumulus-oocyte complexes with a piece of parietal granulosa cells (CGCGs) from early antral follicles. (a) A COCG was embedded in a collagen gel and cultured in hypoxanthine-free medium for 8 days. Granulosa cells spread into collagen gel. (b) A COCG cultured in 4 mM hypoxanthine-supplemented medium for 8 days. The COCG formed an antrum-like structure. Scale bar represents 500 pm. Table 1. Effect of hypoxanthine on the growth of bovine oocytes from early antral follicles Before

culture

After

culture

Hypoxanthine (mM)

No. of COCGs cultureda

Mean diameter of oocytes f SD km)

0

33

95.3 zt 2.8b

14 (42)b

107.1 f 8.3b*

2

29

95.2 f 3.Ob

14 (48)bc

109.1 f 9.Ob*

4

33

94.9 f 2.8b

23 (70)~

111.7 f 9.7b”

No. of oocytes showing normal morphology (%)

Mean diameter of oocytes f SD @m)

a Oocytes enclosed with cumulus cells with a piece of parietal granulosa cehs (CoCGs) were embedded in collagen gels, and cultured in the medium containing 10% fetal calf serum, 0.1 mg/ml sodium pyruvate and hypoxanthine (0,2 or 4 mM) for 8 days. b-c Values with different superscripts in the same column differ significantly (RW5). * Each of the means differs significantly from the value before culture (P-&05, Student’s t-test). Effect of FSH on Growth of Bovine Oocytes The COCGs from early antral follicles were cultured in media containing 4 mM hypoxanthine, and 10 or 100 r&ml FSH. After 3 to 4 d of culture, the COCGs formed antrum-like structures. The development of the antrum-like structure was highest in the 10 q/ml FSH group. Of the COCGs, 37 (10/27), 77 (10/26) and 41% (12/29) maintained these antrum-like structures until 8 d of culture in the media supplemented with 0, 10 and 100 ng/ml FSH, respectively. The mean diameters of surviving oocytes increased significantly in all groups compared to values before culture (Table 3). All of the oocytes which had recovered from the antrum-like structures were enclosed-by granulosa

Theriogenology

748

cells, and the number of oocytes surrounded by granulosa cells was significantly higher in the 10 rig/ml FSH-supplemented group than in the others (Table 4). The oocytes surrounded by granulosa cells were at the GV stage. Some of the denuded oocytes by the end of the culture period had undergone GVBD, and 15 (4/13) and 14% Q/7) of the oocytes had resumed meiosis in FSH-free and 100 ng/ml FSH-supplemented media, respectively.

Table 2.

EfIect of hypoxanthine on the nuclear stage of bovine oocytes from early antral follicles after culture

No. of No. (%) of oocytes with GCb xanthine COCGs (n&I) cultureda Total GV D Ml Deg

HYP-

No. (%) of denuded oocytes Total

GV

D

MI

Deg

0

33

1 (3)c

1 (3)” 0 (0) 0 (0) 0 (0; 32 (97)~ 8 (24)~ 4 (12)~ 1 (3)~ 19 (58)~

2

29

8 (28)d

8 (28)d 0 (0) 0 (0) 0 (0) 21 (72)d 2 (7)“1 4 (14)~ 0 (0) 15 (52)~~’

4

33

15 (45)d 15 (45)6 0 (0) 0 (0) 0 (0) 18 (55)d 1 (3)d 5 (15)~ 2 (4)~ 10 (3O)d

a Cktcytes enclosed with cumulus cells with a piece of parietal granulosa ceils (COCGs) were embedded in collagen gels, and cultured in the medium containing 10% fetal calf serum, 0.1 mglml sodium pyruvate and hypoxanthine (0,2 or 4 mM) for 8 days. b GC: granulosa cells, GV: germinal vesicle stage, D: diakinesis, MI: metaphase I, Deg: degenerated. c.d Values with different superscripts in the same column differ significantly (Pd.05, x*-test).

Table 3.

Effect of FSH on the growth of bovine oocytes from early antral follicles culture

After

No. of COCGs cultureda

Mean diameter of oocytes * SD @m)

No. of oocytes showing normal morphology (%)

0

27

94.7 f 2.8b

23 (85)b

110.4 f 6.9b*

10

26

94.9 r 3.2b

23 (88)b

107.2 i 6.3bc*

100

29

94.6 f 3.Ob

19 (66)b

106.5 f 4.2c*

Before FSH (ngW

culture Mean diameter of oocytes f SD @m)

a Oocytes enclosed with cumulus cells with a piece of parietal granulosa cells (COCGs) were embedded in collagen gels, and cultured in the medium containing 10% fetal calf serum, 0.1 mg/ml sodium pyruvate, 4 mM hypoxanthine and FSH (0,lO or 100 ng/ml) for 8 days. b.c Values with different superscripts in the same column differ significantly (Pd.05). * Each of the means differs significantly from the value before culture (P&05, Student’s t-test).

Theriogenology

Table 4.

749

Effect of FSH on the nuclear stage of bovine oocytes from early antral follicles after culture

No.of FSH COCGs (@ml) cultureda

No. (%) of oocytes with GCb Total

GV

D

MI

Deg

No. (%) of denuded oocytes Total

GV

D

MI

Deg

0

27

10 (37)c IO (37)c 0 (0) 0 (0) 0 (0) 17 (63)c 11 (41)c 2 (7) 0 (0)

4 (15)c

10

26

20 (77)d 20 (77)d 0 (0) 0 (0) 0 (0)

3 (12)~

100

29

12 (41)~ 12 (41)~ 0 (0) 0 (0) 0 (0) 17 (59)~ 6 (21)d O(0)

a

b

c.d

6 (23)d 3 (12)d 0 (0) 0 (0)

I (3) 10 (34)~

Oocytes enclosed with cumulus cells with a piece of parietal granulosa cells (COCGs) were embedded in collagen gels, and cultured in the medium containing 10% fetal calf serum, 0.1 mg/ml sodium pyruvate, 4 mM hypoxanthine and FSH (0,10 or 100 n&l) for 8 days. GC: granulosa cells, GV: germinal vesicle stage, D: diakinesis, MI: metaphase I, Deg: degenerated. Values with different superscripts in the same column differ significantly (P&.05, x*-test).

MaNrational Competence of In Vitro Grown Oocyte To assess the maturational competence of in vitro-grown bovine oocytes, OCCGs from early antral follicles (Figure 2a) were cultured for 7 and 11 d in the medium containing 4 mM hypoxanthine and 10 ng/ml FSH. Subsequently, the grown oocytes were cultured for 24 h for maturation. The COCGs formed antrum-like structures, and nearly half of them maintained the structure after 11 d in this medium (Figure Zb). Granulosa cell-enclosed oocytes were recovered from the an&urn-like structures (Figure 2~). The mean diameters of the oocytes were 107.5 & 6.1 and 108.0 * 5.3 pm after 7 and 11 d of culture, respectively (Table 5). These diameters were significantly larger than those of the COCGs before Culture, although no difference was observed between those measured after 7 and 11 d. The nuclear morphology of the oocytes enclosed by granulosa cells (mean diameter: 111.3 rt 5.2 pm; n=l5) was examined after 11 d of culture. Most of the oocytes (14/15) were at the GV stage, although only one oocyte had resumed meiosis. The configuration of the chromatin and nucleolus in the GV was different from that seen in small oocytes from early antral follicles before culture. Small oocytes from early antral follicles had a GV which contained spreading fine filamentous chromatin and 1-3 aceto-orcein-stained vacuolated nucleolus (Figure 3a). In contrast, in the oocytes grown in vitro chromatin condensed around the nucleolus in 73% (11/15). In addition, the nucleolus in 47% of the oocytes (7/15) was not stained by orcein (Figure 3b). As a control, oocytes from early antral follicles which were 90 to 99 pm in diameter were cultured in the basic medium for maturation. After 24 h, most of the oocytes did not resume meiosis (Table 6). In vitro-grown oocytes enclosed by granulosa cells were further cultured for maturation for 24 h in the same manner to assess their maturational competence. Only 17% (5130) of the oocytes which had been cultured for 7 d resumed meiosis. In contrast, 89% (31135) of the oocytes cultured for 1I d resumed meiosis. Of the oocytes which had grown to 100 to 109 and 110 to 119 pm in diameter, 87 (20/23) and 91% (IO/l 1) underwent GVBD, respectively. In the former group, oocytes reached the metaphase I stage, although no oocytes progressed beyond that stage. In contrast, 36% (4/l 1) of the oocytes grown to 110-l 19 pm reached metaphase II with emission of the first polar body (Figures 3c and 3d).

Theriogenology

Figure 2.

Bovine cumulus-oocyte complexes with a piece of parietal granulosa cells (COCGs) from early an&al follicles before culture (a). The COCGs were embedded in a collagen gel and cultured in 4 mM hypoxanthine- and 10 ng/ml FSH-supplemented medium for 11 days. The COCGs formed an antrum-like structure (b). From the antrum-like structures, granulosa cell-enclosed oocytes were recovered (c). The collagen gel was digested by collagenase in (b). Scale bars in (a) and (c) represent 100 pm, and 500 ym in (b).

Table 5.

Growth of bovine oocytes from early antral follicles cultured for 7 or I I days Before culture

Duration of culture (days)

No. of COCGs cultureda

After

culture

Mean diameter of oocytes f SD (pm)

No. of oocytes with GCb (%)

Mean diameter of oocyte f SD (pm)

7

57

94.5 f 2.9c

30 (53)c

107.5 f 6. Ic*

11

60

95.2 f 2.7~

35 (58)c

108.0 + 5.3c”

a Oocytes enclosed with cumulus cells with a piece of parietal granulosa cells (CGCGs) were embedded in collagen gels, and cultured in the medium containing 10% fetal calf serum, 0. I mg/ml sodium pyruvate, 4 mM hypoxanthine and 10 @ml FSH for 7 or I 1 days. b GC: granulosa cells. c Values with different superscripts in the same column differ significantly (P-&LOS). * Each ofthe means differs significantly from the value before culture (P&05, Student’s t-test).

Filgure

3.

The typical nuclear morphology of bovine oocytes from early arma follicles before culture (a}. Filamentous chromatin distributed throughout the germinal vesicle. Three nucleoli are stained by orcein and contain vacuoles. In oocytes grown in vitro, chromatin has started condensation around a nucleolus with large vacuoles which is not stained by orcein (b), After maturation culture of the oocytes grown in vitro for I I days, some of them matured to metaphase II wNiththe first polar body (c and d). The first polar body (c) and the metaphase spindle (d) were observed at different focuses. All of the oocytes were fixed in acetic ethanol, stained with 1% aceto-orcein and prepared as whole mounts.

2

752

Theriogenology

Table 6. In vitro maturation of bovine oocytes cultured for 7 or 11 days in vitro Duration of culture (days)

Oocyte diametera (pm)

No. of oocytes examined

No. (%) of oocytes resumed meiosisb Total

D

MI

M II

2 (1l)d

2(11P

0 (0)

0 (0)

0 (0)

0 (0)

0 0-J)

3 (15)h 0 (0) 0 (0)

1 (5)d 0 (0)

I (lof3

0 (0) 0 (0) 0 (0) 0 (0)

5 uv

0 (0)

3 (10)d

2 (7)d

0 (0)

0 (0) 19 (83)f 6 (55)ef

0 (0) 0 (0) 4 (36)d

0 (0)

0

90-99

18

7

90-99 loo-109 110-119 120-129

1 20 8 1

Total

30

90-99 loo-109 110-119 120-129

0

0 (0)

0 (0)

23 11 1

20 (87)e 10 (91)e 1000)

I (4)d 0 (0) 0 (0)

Total

35

31 (89)e

1 (3)d

11

No.(%) of oocytes degenetatedc

4 (20)’

0 (0)

1 ww

26 (74)e

I (100)

O(O)

1 t4)d 1 (9)d 0 (0)

4(11)d

2 (7)d

a Oocytes enclosed with cumulus ceils with a piece of parietal granulosa cells were embedded in collagen gels, and cultured in the medium containing 10% fetal calf serum, 0.1 mglml sodium pyruvate, 4 mM hypoxanthine and 10 nglml FSH for 7 or 11 days. After 7 or 11 days of culture, granulosa cell-enclosed oocytes were collected from the complexes, and were cultured in 10% fetal calf serum and 0.1 mg/ml sodium pyruvate supplemented medium for 24 h. b D: diakinesis, MI: metaphase I, M II: metaphase II. c Oocytes with abnormal chromatin configurations were classified as degenerated oocytes. d,e Values with different superscripts in the same column differ significantly (P&05, x2-test). DISCUSSION The collagen gel embedding culture method has been used for growing mouse oocytes in preantral follicles (27). In this cuhure system, the three-dimensional integrity of the follicles is maintained, and granulosa cells proliferate in the follicles. In the pig, preantral follicles embedded in collagen gel maintain their three-dimensional structure, and some of the follicles form an antrum (18). When bovine oocyte-cumulus-granulosa cell complexes (COCGs) collected from early antral follicles were cultured without being embedded in collagen gel in our preliminary experiment, the integrity of the follicles was disrupted and no oocytes survived after 8 d of culture. However, when bovine CGCGs were embedded in coflagen gel and cultured, granuIosa cells ‘surrounded cumulusenclosed oocytes, and CGCGs formed an antrum-like structure in the gels. Furthermore, some oocytes grew to over 110 pm in diameter inside the antrum-like structures. Bovine oocytes have been reported to achieve complete nuclear maturation to metaphase II with a diameter of 110 pm in vivo (13). In the present study, the number of oocytes enclosed by granulosa cells increased significantly when bovine COCGs were cultured in hypoxanthine-supplemented medium. All of the granulosa cell-enclosed oocytes survived and grew to over 100 pm in diameter. It is not clear why hypoxanthine promotes a continued association between oocytes and granulosa cells. Moreover,

Tberiogenology

753

the physiological role of hypoxanthine with respect to the bovine oocyte is obscure because bovine follicular fluid does not contain levels of hypoxanthine comparabIe to that in other animals (8). It has been suggested that the level of CAMP in granulosa cells affects both their development and function (6). Hypoxanthine in the culture medium is thought to maintain CAMP levels in granulosa cells by the inhibitory action on CAMP-phosphodiesterase. It has also been reported that a synthetic CAMP-phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), promotes an association between mouse oocytes and surrounding granulosa cells (10). Surviving bovine oocytes increase in diameter regardless of their adhesion to granulosa cells after culture. Oocytes acquire meiotic competence during the growth phase in the ovaries, and the meiotically competent oocytes approaching their final size resume meiosis spontaneously without hormonal stimulation when they are liberated from the antral follicles. It is thought that oocytes grown in vitro resume meiosis spontaneously. In fact, some of the in vitro-grown and denuded bovine oocytes had undergone GVBD even in the hypoxanthine-supplemented medium, whereas all of the oocytes surrounded by granulosa cells were at the GV stage after growth culture. Hypoxanthine has been reported to inhibit the resumption of meiosis in fully grown mouse and pig oocytes (11, 22). In contrast, fully grown bovine oocytes from ovaries are not as sensitive to hypoxanthine as oocytes from other animals (26). Maturation to metaphase II is inhibited in fully grown bovine oocytes which are enclosed by cumulus cells when they are cultured with granulosa cell monolayers (25). The inhibitory action of the granulosa cells is not observed in the conditioned medium, suggesting that some inhibitory factor in the granulosa cells passes through a junctional association between the granulosa cells and cumulus cehs, reaches the oocytes, and goes into action. It seems likely that hypoxanthine has a role in maintaining an association between the oocyte and surrounding granulosa cells of in vitro-grown bovine oocytes and that the cells exert an inhibitory action on resumption of meiosis in the oocytes through this association. The anti-urn-like structures of the bovine COCGs were well-developed in 10 ng/ml FSHsupplemented medium, although no significant FSH effects were observed in the surviving oocytes. Cultured granulosa cells are morphologically and functionally influenced by FSH (I). When rat granulosa cells in a monolayer are exposed to FSH, they are transformed into a spherical shape and begin to grow in multilayered aggregates while. in culture (1). Rat preantral follicles cultured in FSH-supplemented medium form antrum-like structures with a proliferation of granulosa cells, whereas granulosa cells spread out uniformly around the granulosa cell-enclosed oocytes without FSH (I$ In cultured bovine preantral follicles, FSH promotes follicle development (20, 24, 28). It is thought that, in our experiments, FSH stimulated a proliferation of bovine granulosa cells and maintained morphological quality, consequently allowing the antrumlike structures and the association between the oocytes and the granulosa cells to be maintained. The meiotic competence of the oocytes from early antral follicles was limited. The present study shows that some oocytes acquired maturational competence to metaphase II after the growth culture. After 7 and 11 d of culture, the oocytes grew to 107.5 f 6.2 and 108.0 f 5.3 pm, respectively, although there was no significant difference in diameters. However, the proportions of the oocytes resuming meiosis were significantly different between these 2 groups; 17, and 89% in 7 and 11 d of culture, respectively. The reason for the difference in our experiment is not clear. When naked growing mouse oocytes are co-cultured with fibroblasts or in fibroblast-conditioned medium (2, 3), the oocytes survive and acquire meiotic competence, although they fait to grow. Canipari et al. (2) have suggested that growing mouse oocytes have an autonomous chronological program for acquisition of meiotic competence. Because dibutyryl CAMP and forskoline, an adenyiate cyclase activator, promote acquisition of meiotic competence in naked growing oocytes, Chesnel et al. (3) suggest that mouse oocytes acquire meiotic competence in part according to an autonomous program, but they also suggest that an external stimulation provided by companion somatic cells is required for most of the oocytes to become fully competent with a timing equivalent to that found in vivo (3). It has been estimated that bovine ovarian follicles 0.68 mm in diameter need 6.8 d to reach 3.67 mm, and another 7.8 d is required to reach a preovulatory size of 8.56 mm

754

Theriogenoiogy

(21). In the present study, bovine growing oocytes in the early antral follicles may have acquired meiotic competence according to an autonomous program during the culture period. It appears likely that hypoxantbine and FSH participate in this acquisition of meiotic competence in growing bovine oocytes in vitro. REFERENCES 1. Amsterdam A and Rotmensch S. Structure-function relationships during granulosa cell differentiation. Endocrine Rev 1987;8:309-337. 2. Canipari R, Palombi F, Riminucci M, Mangia F. Early programming of maturation competence in mouse oocytes. Dev Biol 1984;102:519-524. 3. Chesnel F, Wigglesworth K, Eppig JJ. Acquisition of meiotic competence by denuded mouse oocytes: participation of somatic-cell product(s) and CAMP. Dev Biol 1994;161:285-295. 4. Downs SM, Coleman DL, Ward-Bailey PF, Eppig JJ. Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight fraction of porcine follicular fluid. Proc Nat Acad Sci USA 1985;82:454458. 5. Eppig JJ. Mouse oocyte development in vitro with various culture systems. Dev Biol 1977; 60~37 l-388. 6. Eppig JJ. Mammalian oocyte development in vivo and in vitro. In: Wassarman PM (ed), Elements of Mammalian Fertilization. Boca Raton FL: CRC Press, 1991;57-76. 7. Eppig JJ, Downs SM. The effect of hypoxanthine on mouse oocyte growth and development in vitro: maintenance of meiotic arrest and gonadotropin-induced oocyte maturation. Dev Biol 1987;119:313-321. 8. Eppig JJ, Downs SM. The role of purines in the maintenance of meiotic arrest in mammalian oocytes. In: Haseltine FP, First NL (eds), Meiotic Inhibition: Molecular Control of Meiosis. New York: Alan R. Liss Inc, 1988;103-113. 9. Eppig JJ, O’Brien MJ. Development in vitro of mouse oocytes from primordial follicles. Biol Reprod 19%;54: 197-207. 10. Eppig JJ, Schroeder AC. Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro. Biol Reprod 1989; 41:268-276. 11. Eppig JJ, Ward-Bailey PF, Coleman DL. Hypoxanthine and adenosine in murine ovarian follicular fluid: concentrations and activitv in maintaining_ oocvte meiotic arrest. Biol Reurod . 1985;33: 1041-1049. 12. Erickson BH. Development and senescence of the postnatal bovine ovary. J Anim Sci 1%6, 25800-805. 13. Fair T, Hyttel P, Grave T. Bovine oocyte diameter in relation to maturational competence and transcriptional activity. Mol Reprod Dev 1995;42:437-442. 14. Figueiredo JR, Hulshof SCJ, van den Hurk R, Nusgens B, Bevers MM, Ectors FJ, Beckers JF. Preservation of oocyte and granulosa cell morphology in bovine preantral follicles cultured in vitro. Theriogenology 1994;41:1333-1346. 15. Gore-Langton RE, Daniel SAJ. Follicle-stimulating hormone and e&radio1 regulate antrumlike reorganization of granulosa cells in rat preantral follicle cultures. Biol Reprod 1990;43:6572. 16. Gosden RG, Bownes M. Molecular and cellular aspects of oocyte development. In: Grudzinskas JG, Yovich JL (eds), Gametes-The Oocyte. Cambridge: Cambridge University Press, 1995;23-53 17. Gosden RG, Telfer E. Numbers of follicles and oocytes in mammalian ovaries and their allometric relationships. J Zoo1 Land 1987;211:169-175. 18. Hirao Y, Nagai T, Kubo M, Miyano T, Miyake M, Kato S. In vitro growth and maturation of pig oocytes. J Reprod Fertil 1994;100:333-339. 19. Hulshof SCJ, Bevers MM, van der Donk HA, van den Hurk R. The isolation and characterization of preantral follicles from foetal bovine oocytes. 12th Int Cong Anim Reprod, Hague 1992;1:336-338.

Theriogenology

755

20. Huishof SCJ, Figueiredo JR, Beckers JF, Bevers MM, van der Donk JA. van den Hurk K. Effects of fetal bovine serum, FSH and I7 /? -estradiol on the culture of bovine preantral follicles. Theriogenology 1995;%217-226. 21. Lussier JG, Matton P, Dufour JJ. Growth rates of follicles in the ovary of the cow. J Reprod Fertil 1987;81:301-307. 22. Miyano T, Ebihara M, Goto Y, Hirao Y, Nagai T, Kato S. Inhibitory action of hypoxanthinc on meiotic resumption of denuded pig follicular oocytes in vitro, J Exp Zoo1 1995;273:70-75. 23. Motlik J, Koefoed-Johnsen HH, Fulka J. Breakdown of the germinal vesicle in bovine oocytes Cultivated in vitro. J Exp Zoo] 1978;205:377-384. 24. Ralph JH, Wilmut I, Telfer EE. In vitro growth of bovine preantral follicles and the influence of FSH on follicular and oocyte diameters. J Reprod Fertil 1995: 156 abstr. 25. Sirard MA, Bilodeau S. Effects of granulosa cell co-culture on in-vitro meiotic resumption 01‘ bovine oocytes. J Reprod Fertil 1990;89:459465. 26. Sirard MA, First NL. In vitro inhibition of oocyte nuclear maturation in the bovine. Rio1 Reprod 1988;39:229-234. 27. Torrance C, Telfer E, Gosden RG, Quantitative study of the development of isolated mouse pre-antral follicles in collagen gel culture. J Reprod Fertil 1989;87:367-374. 28. Wandji SA, Epigg JJ, Fortune JE. FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles. Theriogenolopy 1996:45:817-832.