Preservation of oocyte and granulosa cell morphology in bovine preantral follicles cultured in vitro

Theriogenology41:1333-1346,1994

PRESERVATION OF OOCYTE AND GRANULOSA CELL MORPHOLOGY IN BOVINE PREANTRAL FOLLICLES CULTURED IN VITRO

J.R. Figueiredo,Iya S.C.J. Hulshof,3 R. Van den Hurk,3 B. Nusgens,2 M.M. BeversP F.J. Ectorsl and J.F. Beckers’

1 Department of Animal Endocrinology and Reproduction Faculty of Vetermary Medicine

2 Laboratory of Experimental Dermatology University of Liege, B-4000 Liege, Belgium

3 Department of Functional Morphology 4 Department of Herd Health and Reproduction

Faculty of Veterinary Medicine, University of Utrecht Yalelaan 8,350s TD, Utrecht, The Netherlands

Received for publication: &ne 30, 1993

Accepted: February 12, 1994

ABSTRACT

Described in the present paper is a culture system that preserves oocyte and granulosa cell morphology in bovine preantral follicles during 5 d in vitro. The effects of additional hypoxantbine and energy substrata (i.e., pyruvate and glutamine) on the morphology of cultured preantml follicles were investigated. It was shown that addition of a mixture of pyruvate, glutamine and hypoxantine to the culture medium increased the percentage of follicles with an intact oocyte from 29.4 to 78.6%.

Morphological criteria are described to discriminate between normal and degenerated preantral follicles during culture by inverted microscopy. In addition, the importance of histological evaluation to judge the quality of oocyte and granulosa cells is demonstrated.

Key words: culture, preantral follicles, substrate, bovine

Acknowledgments

This work was supported by an IRSIA grant from the Faculty of Veterinary Medicine,

The State University of Liege; by FRSM; and by les Actions de Recherches Concert&s de

la Communaute Francaise de Belgique. We thank G. Dijkstra for histology and H.H. Otter, 0. van der Veen and H. Post for

assistance in photography.

aJ.R. Figueiredo is a recipient of a grant from CNPq-BIOAGRO (UFV) of Brazil.

Copyright 0 1994 Butterwotth-Heinemann

1334 Theriogenology

INTRODUCTION

The ability to culture ovarian follicles from the primary stage to the preovulatory stage,

followed by fertilization of their oocytes would have important applications in animal breeding

and human in vitro fertilization programs (4, 40). Exploitation of this resource, however,

requires the establishment of protocols for their growth and development that would promote

and maintain competence for fertilization and embryogenesis.

In mice (8, 9, 17), pigs (27) rats (12) and cats (30) isolated preantral follicles were

cultured and growth of the follicles was observed. Antral follicle development from preantral

follicles in culture has been reported in mice (37,40). Complete development of murine primary

follicles to mature stages was obtained and development to term achieved when oocytes derived

from in vitro-grown follicles were fertilized in vitro (20). Several culture media were used in the

different experiments (8, 12, 17,34,35,40,45). Pyruvate (8, 12, 17, 20, 45) and glutamine

(8, 37) have been used as energy substrate for the culture of ovarian preantral follicles in rats

and mice. Hypoxantbine has been added to the culture medium to prevent meiotic resumption

of the oocyte in several species (15, 19, 47). In addition, Eppig et al. (19) showed that

hypoxanthine reduces the percentage of denuded oocytes in mu&e preantral follicles cultured in

vitro.

Maintaining oocyte viability in preantral follicles has been shown to be difficult. Maresh et

al. (35) reported that in rabbit preantral follicles all oocytes were degenerated after a 15-d culture

period. Hirao et al. (27) reported that in pig pmantral follicles the survival rate of oocytes

cultured in medium with or without 10 mg/ml FSH was 36 and 15%, respectively. In the cow,

Jewgenow et al. (29) showed that after 48 h of culture, most of the oocytes in preantral follicles

were degenerated. Recently, Nuttinck et al. (38) found that 85% of the preantral bovine follicles

remained viable during a 7-d culture period on irradiated fibroblasts. It is, however, unclear

whether oocytes were present in those follicles.

The lack of efficient methods for the isolation of preantral follicles from the ovaries as

well as of defined culture systems has restricted studies in follicular viability and growth.

Recently, we reported on mechanical and enzymatic methods for the dissociation of bovine

ovaries to yield large numbers of intact, small preantral follicles (22). The present paper deals

with the culture of isolated bovine preantral follicles over a 5-d period, with emphasis on the

(micro)morphological condition of granulosa cells and oocytes, and on the effects of pyruvate,

glutamine and hypoxanthine.

Theriogenology 1335

MATERIALS AND METHODS

Isolation of Preantral Follicle

Ovaries from adult cows were collected at a local slaughterhouse. The ovaries were

aseptically removed, stripped of surrounding fat tissue and ligaments, and transferred into 10 ml

PBS supplemented with 200 IU/ml of penicillin and 200 pg/ml of streptomycin. Following 3

successive washings in this solution, each ovary was transferred into 10 ml of Ml99 HEPES

containing 200 IU/ml of penicillin, 200 @ml of streptomycin, and 5% fetal calf serum (KS,

Boehringer Mannheim, Germany) and transported to the laboratory within 1 h in a thermos

flask tilled with water at 39°C.

Preantral follicles were isolated from the ovaries by applying the simple mechanical

procedure described by Figueiredo et al. (22) without the use of enzymes. Briefly, the ovaries

were cut into small fragments using a tissue chopper. The ovarian fragments were put in Ml99

HEPES supplemented with 5% FCS, antibiotics (200 III/ml of penicillin, 200 pglml of

streptomycin), and 0.23mM pyruvate and then suspended 40 times with a pasteur pipette and filtered successively through 500- and lOO-ym nylon mesh filters. Isolated preantral follicles

(30 to 70 ym in diameter) were selected using a mouth-operated micropipette. Groups of 3

follicles were placed in 5@-pl droplets of isolation medium to facilitate the onset of culture. The

total procedure (isolation, selection and manipulation of follicles before culture) took approximately 3 h.

General culture procedure

Isolated preantral follicles were placed in 350 pl of culture medium in groups of 3 follicles

per well (plastic petri dishes, Nunc) and cultured for 5 d at 39’ C in 5% CO, in air. Culture in

groups of 3 follicles per well and their localization after attachment at Day 1 of culture allows

each follicle to be studied individually during the culture period.

The control medium was alpha Minimal Essential Medium (MEM; Gibco-BRL, Geithersburg, MD, USA) supplemented with antibiotics (200 III/ml of penicillin, 200 pglml of

streptomycin, 10% FCS and ITS (Insulin 6.25 pglml, transferin 6.25 pg/ml and selenium 6.25

nglml). Fresh medium was prepared immediately before use in culture.

Culture Experiments

A total of 341 follicles was cultured for 5 d in 8 different culture media. The media were compared using preantral follicles from the same ovaries. Medium was changed 24 h after the

start of culture, and then every other day.

1336 Theriogenology

The media were:

Glu: Hyp: PyrGlu:

PyrHyP GluHyp: PyrGluHyp:

Control medium Control medium plus 0.23 mM pyruvate (Sigma); Control medium plus 2 mM glutamine (Sigma); Control medium plus 2 mM hypoxanthine (Sigma); Control medium plus 0.23 mM pyruvate and 2 mM glutamine; Control medium plus 0.23 mM pyruvate and 2 mM hypoxanthine; Control medium plus 2 mM glutamine and 2 mM hypoxanthine; Control medium plus 0.23 mM pyruvate, 2 mM glutamine and 2 mM hypoxanthine.

Fohicular Morphology and Histological Evaluation

The morphology of each follicle during culture was observed using an inverted microscope. Follicular morphology, particularly the clarity of the oocyte and surrounding granulosa cell layer, was recorded at Days 1 and 5 of culture. Simultaneously, follicular diameters were measured with an ocular micrometer.

After 5 d of culture, the follicles were fmed in Bouins fluid for 24 h, dehydrated in a graded series of ethanol, and embedded in paraffin wax. Then 5ym tissue sections were stained with PAS haematoxylin. Freshly isolated preantral follicles were also fixed and sectioned to compare the in vivo appearance of preantral follicles with the in vitro picture.

The number of cells per mm2 in the granulosa layer was measured in 20 sections of uncultured preantral follicles, and of follicles cultured for 5 d in medium with or without the addition of hypoxanthine. In the same 20 sections, the area of 50 granulosa cell nuclei was

measured. The number of cells per mm2 in the granulosa layer and the area of the nuclei were determined by means of image analysis using the TIM image processing program (Version 3.20, Difa Measuring systems, Breda, The Netherlands) and a black and white CCD video camera (MO- Type High Technology Holland, Eindhoven, The Netherlands).

Statistical Analysis of Data

The percentages of morphologically normal follicles after 24 h (Day 1) of culture and after an additional 4 d of culture (Day 5) were analyzed by Chi-square. Since follicles were monitored individually during culture, the increase in diameter of morphologically normal follicles from Day 1 to Day 5 was analyzed by t-test (analysis of variance) and were considered paired data. The number of cells per mm2 in the granulosa cell layer and the area of the granulosa cell nuclei were analyzed by the nonparametric Mann Whitney U test. Values were considered statistically significant when PcO.05.

Theriogenology

RESULTS

1337

Selection of Bovine Preatmal Follicles Before Culture

After the isolation procedure, the remaining suspension was studied under an inverted

microscope and appeared to contain numerous morphologically normal follicles, dark follicles

and denuded oocytes. The morphologically normal follicles (Figure la) showed a basal lamina

and no apparent sign of degeneration. Histological evaluation revealed that normal follicles were

composed of an intact oocyte, surrounded by 1 to 2 layers of healthy granulosa cells, and an

intact basal lamina (Figure lb). Dark follicles (Figure lc) contained large vacuoles and they did

not attach to the substratum when put in culture. Therefore, in subsequent in vitro experiments

only morphologically normal follicles were used.

Figure 1. Freshly isolated a, b) morphologically normal and c) dark preantral follicles

from cow ovaries. a, c) inverted microscope (x 160); b) histological section (x 600).

Morphology of Cultured Bovine Freantral Follicles

During culture, the preantral follicles attached to the plastic substratum and retracted,

making visible the oocytes in most of the primary and in some of the secondary follicles.

Spreading of the gmnulosa cells was not observed. Based on their morphology under inverted

microscopy, the cultured follicles were allocated into 3 classes: morphologically normal follicles, with a clear oocyte surrounded by a clear granulosa layer (Figure 2a); degenerated

Type 1, with a dark, contracted oocyte surrounded by a clear granulosa layer (Figure 2~); and

Theriogenology

degenerated Type 2, with a complete dark appearance of the follicle (Figure 2e). Histological evaluation of these 3 classes of follicles revealed that in all types a surrounding basal lamina was

present. Morphologically normal follicles showed an intact oocyte surrounded by 1 or 2 layers

of granulosa cells without pycnotic nuclei or mitotic figures (Figure 2b). In the degenerated

Type 1 follicles, the contracted oocytes stained strongly with Periodic Acid Schiff; the nuclear

chromatin was also clumped (Figure 2d). In degenerated Type 2 follicles, no oocyte was

present (Figure 20. While the granulosa cells of degenerated Type 1 follicles were apparently

unaffected, pycnotic nuclei occasionally appeared in the granulosa of degenerated Type 2

follicles.

Figure 2. Morphology of cultured bovine preantral follicles: a, b) morphologically normal follicle, c, d) degenerated Type 1 follicle, and e, f) degenerated Type 2 follicle.(a, c, e: inverted microscope (x 480); b, d, f: histological section (x 600).

During a further 4 d of culture, it was observed that morphologically normal follicles either remained morphologically normal or evolved into degenerated Type 1 or degenerated Type 2. Degenerated Type 1 follicles remained degenerated Type 1 or evolved into degenerated Type 2, whereas degenerated Type 2 always remained degenerated Type 2. In all 3 classes of follicles, approximately 45% of the follicles had detached during culture and were lost in the changing of the medium. Detachment thus occurred independently of follicular morphology, and it greatly reduced the number of follicles present after 5 d of culture.

Theriogenology

Effect of Pyruvate, Glutamine and Hypoxanthine on the Percentage of Morphologically Normal

Follicles During Culture

Table 1 shows the numbers and percentages of morphologically normal follicles at Day 1

and Day 5 of culture in 8 different media. At Day 1, a higher percentage of morphologically

normal follicles was observed in medium supplemented with a mixture of pyruvate, glutamine

and hypoxanthine (PcO.01) or a mixture of hypoxanthine and glutamine (PcO.05) compared

with the control. Other mixtures (pyruvate plus glutamine and pyruvate plus hypoxanthine) or

supplements alone (pyruvate, glutamine and hypoxanthine) did not significantly increase the

percentage of morphologically normal follicles compared with the control.

To evaluate the capacity of different culture media in preserving normal folhcular

morphology during an additional 4 d of culture, morphologically normal follicles presented at

Day 1 were individually reevaluated at Day 5 (Table 1). During this period, 36.2 to 62.5% of

the morphologically normal follicles had detached from the plastic substrate and were lost

during changing of the medium. This explains the difference between the number of follicles

classified as morphologically normal follicles at Day 1 of culture and the number available to

calculate the percentages of morphologically normal follicles at Day 5 (e.g., for the control

group; of the 28 follicles classified as morphologically normal follicles at Day 1 of culture, 11

had detached and were lost during the changing of medium, whereas 17 follicles were still

attached. of these 17 follicles, 5 (29.4%) remained morphologically normal follicles). During

an additional 4 d of culture (Day 5), the highest percentage of morphologically normal follicles

was observed when control medium was supplemented with a mixture of pyruvate, glutamine

and hypoxanthine. Addition of pyruvate, glutamine and hypoxanthine alone and mixtures of

pyruvate plus glutamine, pyruvate plus hypoxanthine, and glutamine plus hypoxanthine

increased the percentage of morphologically normal follicles compared with that of the wntrol

group, but the differences were not statistically significant_

Figure 3 shows the mean diameter f SEM of morphologically normal follicles at Day 1

and Day 5 of culture. Mean diameters of morphologically normal follicles significantly increased

during culture in all the media tested (PcO.05) excepted for the medium suplemented with a

mixture of pyruvate and glutamine (PsO.05). The mean diameters among the different media

were not statistically different, either at Day 1 or at Day 5 (PrO.05). Larger variations in the

increase of follicular diameter were observed among follicles cultured in the absence of

hypoxanthine. Furthermore, the area of the granulosa cell nuclei was larger and the number of

cells per mm2 in the granulosa layer was lower in follicles cultured for 5 d in the absence of

hypoxanthine than in the uncultured follicles or in follicles cultured in the presence of hypoxanthine (Table 2).

Tab

le 1

. N

umbe

r (n)

and

per

cent

age (

betw

een p

aren

thes

es) o

f mor

phol

ogic

ally

norm

al fo

llicl

es (M

NP)

afte

r 1

day

(Day

1) a

nd a

fter

an

z

addi

tiona

l 4 d

ays o

f cul

ture

(Day

5) i

n m

edia

with

or w

ithou

t add

ition

al py

ruva

te (P

), g

luta

min

e (G

) and

/or h

ypox

anth

ine (

H).

$

Con

trol

P

%ul

ture

Med

ium

G

H

PG

PH

G

H

PGH

Day

1

ntot

al

n M

NF

(%)

MN

PatD

ay 1

deta

ched

from

day

1

toda

y5

Day

5

n le

ft

n M

NF

(%)

42

41

32

46

40

58

41

41

28(6

6.6)

26

(63.

4)

19(5

9.3)

34

(73.

9)

24(6

0.0)

47

(81.

0)

35(8

5.3)

* 37

(90.

2)**

11

13

7 14

15

17

14

17

13

12

20

9 30

21

5(29

.4)

6(46

.1)

5(41

.6)

12(6

0.0)

3(

33.3

) 15

(50.

0)

12(5

7.1)

n le

ft =

num

ber o

f MN

P at

Day

1 m

inus

the

num

ber o

f MN

F d

etac

hed

from

Day

1 to

Day

5.

*P=

< 0

.05

and

**P=

< 0.

01 in

dica

te si

gnif

ican

t dif

fere

nces

com

pare

d w

ith th

e co

ntro

l. 9 0 0‘

23

14

5r

1 l(7

8.6)

” 5.

3

_ __

__

___.

__

-.

.-

. ._

-

_I

-.

__

_~

-.

- -

- -

. -

Theriogenology 1341

ij 40 .C( a Li 2f9 m ‘3 .Y 0 = g

13 P G PG PH GH ~GH Medium 5 6 5 lH2 3 15 12 11 u

Figure 3. Mean diameters f SEW of morphologically normal follicles in 8 different media.

n= Number of morphologically normal follicles that remained attached from Day 1 to Day 5 of culture.

* (P ~0.05) indicate significant differences between Day 1 and Day 5 of culture

in each treatment. Paired data were used since individual follicles were

measured again at Day 5.

Table 2. Number of cells per mm2 in the granulosa layer and area of granulosa cell nuclei in uncultured prea.ntral follicles, and in preantral follicles cultured for 5 d in the presence or absence of hypoxanthine.

Number of cells per mm2

granulosa (mean + SD)

Area of granulosa cell

nuclei in pm2 (mean f SD)

Uncultured follicles Absence of hypoxanthine Presence of hypoxanthine

14.1 f 3.7 27.51 f 3.3

9.4 f 2.2* 34.75 f. 5.9*

12.9 rt 2.9 26.37 + 3.6

* Values are significantly different, Mann Whitney U-test (PC 0.05).

DISCUSSION

Maintenance of oocyte viability is the major problem in the culture of pmantral follicles (27,30,35). The degeneration signs observed during culture were those of contraction of the oocyte, condensation of the nucleus, and increased PAS staining of the cytoplasm. This was

1342 Theriogenology

followed by complete disappearance of the oocyte. These degenerative changes correspond with those described for atretic preantral follicles in vivo (6). In preliminary unpublished experiments using bovine follicles, we observed that follicle viability appeared to be negatively

affected by excessive manipulation of preantral follicles before culture, by the duration of time

(more than 4 h) between the beginning of the isolation procedure and the onset of culture, and

by the composition of the isolation medium. When PBS was used as the isolation medium,

most follicles were degenerated Type 1 follicles after 1 d of culture.

The present study showed that addition of pyruvate, glutamine or hypoxanthine to the

culture medium increased the percentage of follicles with an intact oocyte from 29.4 to 78.6 %.

Maintenance of the oocyte in almost 30 % of the follicles cultured in control medium may be due

to the presence of pyruvate in MEM. Previously it was shown that pyruvate was the

predominant substrate used by immature and mature bovine oocytes and zygotes (32). In

addition, Eppig (16) reported that isolated growing mouse oocytes utilized exogeneously

administered pyruvate more efficiently than glucose, lactate or succinate. Several authors (2, 3,

11, 43) have shown that glutamine is an efficient energy substrate for bovine and mouse

embryos, and for hamster and rabbit oocytes. Eppig et al. (19) demonstrated that hypoxanthine

promoted the association of oocytes and accompanying granulosa cells during the culture of

mouse preantral follicles; approximately 70 % of the oocytes became spontaneously denuded of

their granulosa cells when cultured in medium without hypoxanthine. The maintenance of

oocyte-granulosa cell communication via gap junctions has been shown to promote oocyte

growth in vitro (5). More than 85% of the metabolites of the oocytes are transported from the

granulosa cells to the oocyte via gap junctions (26). We showed that micromorphological

appearance of follicles cultured in the presence of hypoxanthine was similar to that of uncultured

follicles. It is probable that under our conditions, hypoxanthine improves the utilization of additional energy substrata by maintaining interactions between the oocyte and the surrounding

granulosa cells. In addition, hypoxanthine has been used in vitro to prevent the resumption of

meiosis in oocytes of different species (14), although a limited inhibitory effect has been reported in bovine oocytes (41). Oocytes acquire meiotic competence when they reach 80% of

their !dnal size (44). In the bovine species, this occurs when the follicles reach the antral stage

(i.e., diameter larger than 3 mm; 42). Consequently, the oocytes enclosed in our preantral

follicles used in our present study were far from acquiring meiotic competence.

Morphological examination using the inverted microscope has been shown to be very useful for the evaluation of preantral follicles in vitro. However, histological evaluation has been shown indispensable for the identification of an intact oocyte and to judge the quality of the

gmnulosa cells. Enlargement of preantral follicles after culture in all tested media can only be understood after histological examination. Increase in follicle diameter may be caused by

increase in oocyte diameter and by proliferation of the granulosa cells. Oocyte diameter is difficult to follow during culture, primarily in secondary follicles, due to the limited visibility of

Theriogenology 1343

the oocytes under an inverted microscope. proliferation of the granulosa cells should be tested

in future experiments by adding 3H-thymidine (28) or by bromo-deoxy-uridine (31) to the

culture medium followed by autoradiography or immunohistochemistry. Increase in diameter

may be also be due to enlargement of the granulosa cells, as indicated by the lower number of

cells per mm2 in the granulosa layer, and observed in follicles cultured in medium without

hypoxanthine. The increase in size of the granulosa cells and their nuclei resembles the

differentiation of granulosa cells in response to LH/hCG (1, 10, 13), and thus probably reflects

abnormal follicular development. In follicles cultured with a mixture of pyruvate, glutamine and

hypoxanthine, the appearance of the granulosa cells corresponds with that of uncultured

follicles. The increase in follicular diameter in these latter follicles may, therefore, be due to

proliferation of the granulosa cells.

We observed that 36.2 to 62.5 % of the morphologically normal follicles present at Day 1

of culture later detached from the plastic substratum and were lost during changing of the

medium. Loss of follicles can be attributed to the culture method used and not to the

degeneration of follicles since morphologically normal, degenerated Type 1, and degenerated

Type 2 follicles were lost to the same extent. It is known that attachment of cells to the

substratum may be affected by several factors such as temperature (24, 46) and pH, the

presence of C!a+2 and Mg+2, the concentration of serum (33), the type of cell (21, 36) and

adhesive proteins such as fibronectin (23,25). In culture, cells attach to the substratum either by

producing their own attachment factor or by binding to the attachment factors absorbed on the

substratum or those present in the culture medium (39). Fetal calf serum promotes the

attachment of many cell types in vitro by providing attachment factors (33), although in the

present study, addition of 10% FCS to the culture medium did not prevent follicular

detachment. Follicles probably attach to the substratum by synthesizing attachment factors.

Carnegie (7) reported that the secretion of fibronectin by rat granulosa cells occurs during early follicular development. When dark follicles were placed in culture, they did not attach to the

substratum and are apparently not able to produce attachment factors.

In our present study, granulosa cells did not spread away from the oocytes during culture.

In other studies (18, 35), spreading of granulosa cells from the oocytes was observed when preantral follicles were cultured. These preantral follicles were obtained after enzymatic

digestion with collagenase or trypsin, and it was reported that they were not surrounded by a

basal lamina. Using a mild mechanical isolation procedure, the basal lamina was preserved, and this may have prevented the spreading of granulosa cells in culture and consequently maintained

follicle structure in vitro.

In summary, we conclude that small bovine preantral follicles can be cultured in vitro for

5 d in a medium containing pyruvate, glutamine and hypoxanthine. Furthermore, we have

presented morphological criteria for cultured normal and degenerated follicles and showed that histological examination is indispensable for evaluating the quality of cultured follicles.

1344 Theriogenology

REFERENCES

1. Amsterdam A, Rothemensch S. Structure-function relationships during granulosa cell differentiation.

Endocr Rev 1987;8: 309337. 2. Bae M, Foote RH. Carbohydrate and aminoacids requirements and ammonia production of rabbit

follicularoocytes in vitro. Expl Cell Res 1991;91: 113-l 18. 3. Bavister BD, Leibfried ML, Lieberman G. Development of preimplantation embryos of golden hamster

in a defined culture medium. Biol Reprod 1983;28: 235-247. 4. Betteridge RI, Smith C, Stubbings RB, Xu XP, Ring WA. Potential genetic improvement of cattle by

fertilization of fetal oocytes in vitro. J Reprod Fertil 1989;38 (Suppl.): 87-98. 5. Buccione R, Schroeder AC, Eppig JJ. Interactions between somatic cells and germ cells throughout

mammalian oogenesis. Biol Reprod 199@,43: 543-547. 6. Byskov AG. Follicular atresia. In: Jones, R.E. (ed) The vertebrate ovary, plena in press, New York,

1978,533-562. 7. Carnegie JA. Secretion of fibronectin by rat granulosa cells occurs primarily during early follicular

development. J Reprod Fertil 1991;89: 579-589. 8. Carroll J, Whittingham DG, Wood MJ. Effect of dibutyryl cyclic adenosme monophosphate on granulosa

cell proliferation, oocyte growth and meiotic maturation in isolated mouse primary ovarian follicles cultured in collagen gels. J Reprod fertil 199 1;92: 197-207.

9. Carroll J, Whittingham DG, Wood MJ. Effect of gonadotrophin environment on growth and development of isolated mouse primary ovarian follicles. J Reprod Fertii 1991;93: 71-79.

10. Cavender JL, Murdoch WJ. Morphological studies of microcirculatory system of periovulatory ovine follicles. Biol Reprod 1989;41: 309-316.

11. Chatot CL, Tasca RJ, Ziomek CA. Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium. J Reprod Fertil1990;89: 335-346.

12. Daniel AJ, Armstrong DT, Gore-langton RE. Growth and development of rat oocyte in vitro. Gam Res 1989;24: 109-121.

13. Dieleman SJ, Kruip TAM, Fontijne P, De Jong WHR, van der Weyden GC. Changes in oestradiol, progesterone and testosterone concentration in foll@rlar fluid and in the micromorphology of preovulatory bovine follicles relative to the peak of luteinizing hormone. J Endocrinol1983;97: 3 l-42.

14. Downs SM. Factors affecting the resumption of meiotic maturation in mammalian oocytes. Theriogenology 1993;39: 65-69.

15. Downs SM, Coleman DL, Ward-Bailey PF, Eppig JJ. Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight traction of porcine follicular fluid. Proc Nat1 Acad Sci USA 1985;82: 454-458.

16. EppigJJ. Analysis of mouse oogenesis in vitro. Oocyte isolation and the utilization of exogenous energy sources by growing oocytes. J Bxp Zoo1 1976;198: 375-382.

17. Eppig JJ. Mouse oozyte development in vitro with various culture systems. Dev Biol 1977;60: 371-378. 18. Eppig JJ. Growth and development of mammalian oocytes in vitro. Arch Path01 Lab Med 1992; 116: 379-

382. 19. Eppig J, 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. 20. Eppig JJ, Schoeder AC. Capacity of mouse oocyte t?om preanttal follicles undergoes embryogenesis and

development to live young after growth, maturation, and fertilization in vitro. Biol Reprod 1989;41: 268- 276.

2 1. Elsale T, Bard J. Collagen substrata for studies on cell behavior. J Cell Biol 1972;54: 626-637.

Theriogenology 1345

22. Figueiredo JR, Hulshof SCJ, Van den Hurk R, Ectors FJ, Fontes RS, Nusgens B, Bevers MM, Beckers JF. Development of a new mechanical and enzymatic method for the isolation of intact preanlral follicles from fetal, calf and adult bovine ovaries. Theriogenology 1993;40: 789-799.

23. Gold LI, Pearlstein E. Fibronectin-Collagen binding and requirement during cellular adhesion. B&hem J 1980;186: 551-559.

24. Goldberg B. Binding of soluble type I collagen molecules to the fibroblast plasma membrane. Cell 1979;16: 265-275.

25. Grinnell F, Feld MK. Initial adhesion of human fibroblasts in serum free-medium: Possible role of secreted fibronectin. Cell 1979;17: 117-129.

26. Heller D, Cahill D, Schultz RM. Biochemical studies of mammalian oogenesis: metabolic cooperativity between gmnulosa and growing oocytes. Dev Biol 1981;84: 455-464.

27. Hirao Y, Miyano T, Kato S. In vitro growth of porcine oocytes. 12th Int. Cong Anim Reprod 1992;l: 333-335.

28. Hirshifield AN. Granulosa cell proliferation in very small follicles of cycling rats studied by long-term continuous tritiated-thymidme infusion. Biol Reprod 1989;41: 309-316.

29. Jewgenow K, Pitra C. Die Isolierung van Praamralfolikeln aus EierstGcken des Rindes. Reprod Dom Anim 1991;26: 281-289.

30. Jewgenow K, Pitra C. Hormone-contiolled culture of secondary follicles of domestic cats. Theriogenology 1993;39: 527-535.

3 1. Kant HJG. Periodic acid incubation can be relate hydrochloric acid hydrolysis and trypsin digestion in immunogold-silver staing of bromo-deoxy-uridine incorporation in plastic sections and allows the PAS reaction. Histochem J 1992;24: 170-175.

32. Khurana N, Niemann H. Energy metabolism of bovine oocytes matured and fertilized in vitro. 12th Int. Cong Anim Reprod 1992;l: 348-350.

33. Klebe RJ. Isolation of a collagen-dependent cell attachment factor. Nature 1974;250: 248-251. 34. Leichthammer F, Baunack E, Brem G. Behavior of living primordial germ cells of livestock in vitro.

Theriogenology 1990;33: 1221-1230. 35. Maresh G A, Timmons TT, Dunbar BD. Effect of matrix on the expression of specific ovarian proteins.

Biol Reprod 1990;43: 965-976. 36. Murray JC, Liotta L, Rennard SI, Martin GR. Adhesion characteristics of murine me&static and

nonmetastatic tumor cells in vitro. Cancer Res 1979;40: 347-35 1. 37. Nayudu PL, Osbom SM. Factors influencing the rate of preantml and antral growth of mouse ovarian

follicles in vitro. J Reprod Fertil 1992;95: 349-362. 38. Nuttinck F, Mermillod P, Massip A, Dessy F. Characterization of in vitro growth of bovine preantral

follicles: A preliminary study. Theriogenology 1993;39: 81 l- 821.

39. Pearlsten E. Substrata activation of the cell adhesion factor as prerequisite for cell attachment. Int J Cancer 1978;22: 32-35.

40. Qvist R, Blackwell LF, Boume H, Brown JB. Development of mouse ovarian follicles from primary to ovulatory stage in vitro. J Reprod Fertil 1990: 89: 169-180.

4 1. Sirard MA. ~Temporary inhibition of in vitro meiotic resumption by adenylate cyclase stimulation in immanture bovine oocytes. Theriogenology 1990;33: 757-767.

42. Szijllijsi D. Maturation de l’ovocyte. In: Thibault, C. et Ievasseur, M.C. (ed) Ia reproduction chez les mammif&s et l’homme, INRA-Ellipses, 1991,299-314.

43. Takahashi Y, First NL. In vitro development of bovine one cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 1992;37: 963-968.

Theriogenology

44. Thibault C, Sziilliisi D, G&ard M. Mammalian oocyte maturation. Reprod Nutr D&elop 198727: 865 896

45. Torrance C, Telfer E?, Gosden RG. Quantitative study of development of isolated preantral follicles in collagen gel culture. _I Reprod Fertil 1990;87: 367-374.

46. UedaMJy Ito T, Oka& TS, Ohnishi iA. Correlation between membrane fluidity and the critical temperature for cell adhesion. J Cell BioI 197671: 67@674.

47. Warikoo PK, Bavister BD. Hypoxanthine and cyclic adenosine 5’-monophosphate maintain meiotic arrest of rhesus monkey oocytes in vitro. Fertil Steril1989;51: 886-889.