Evaluation of fluids from cystic follicles for in vitro maturation and fertilization of bovine oocytes

ELSEVIER

EVALUATION OF FLUIDS FROM CYSTIC FOLLICLES FOR IN VITRO MATURATION AND FERTILIZATION OF BOVINE OOCYTES M. Takagi,ta Y.H. Choi,] H. KamishitaJ M. Ohtani,2 T.J. Acosta, t M.P.B.Wijayagunawardane, I A. Miyamoto,3 K. Miyazawa, I K. Satol and E. Sato4 ILaboratory of Theriogenology, 2University Farm, 3Department of Animal Science, Obihiro University of Agriculture and Veterinary Medicine, lnada-cho, Obihiro City, Hokkaido 080, Japan, 4Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan Received for publication: May 28, 1997 Accepted: November 5, 1997 ABSTRACT Follicular cysts are defined as cystic structures derived from unovulated follicles. The formation of the cysts appears to be related to failure of the oocyte to resume meiosis. The aim of this study was to evaluate in the bovine: 1) the ability of the fluid from cystic follicles to promote in vitro oocyte maturation and fertilization, 2) the predictive value of the morphology of oocytes derived from cystic follicles on the ability of the follicular fluid to promote in vitro maturation / fertilization as well as of the oocytes to undergo maturation and fertilization. In Experiment 1, the ability of fluid from cystic (and normal) follicles from live and slaughtered cows (to promote) in vitro maturation and fertilization of bovine cumulus-oocyte-complexes (COC's) was assessed by cumulus expansion, sperm penetration, male pronucleus formation and polyspermy rates. Concentrations of progesterone (P4) and estradiol-17~ (E2) were measured in the fluid from cystic follicles collected from live and slaughtered cows. In Experiment 2, we investigated the relationship of the morphology of COC's from cystic follicles, and the effect of the follicular fluids on oocyte maturation as well as P4 and E2 concentrations. In Experiment 1, although sperm penetration and male pronucleus formation were inhibited significantly by fluid from some cystic follicles collected from live and slaughtered cows, there were no significant differences in sperm penetration, male pronucleus formation and polyspermy rates between fluid from cystic follicles collected from live cows, from slaughtered cows and from control groups, regardless of the P4/E2 ratio. In Experiment 2, the morphology of cumulus-oocyte complexes from cystic follicles varied and the pronuclens formation of oocytes after in vitro fertilization was abnormal. On the other hand, the male pronucleus formation rates were not significantly different between the cystic follicular fluids and control, regardless of the P4/E2 ratio. The results of this study suggest that many of the bovine follicular fluids from cystic follicles possess the ability to induce cumulus expansion, nuclear maturation and male pronucleus formation following in vitro maturation and fertilization of bovine oocytes. The morphology of the cumulus-oocytes complexes from cystic follicles seems not to relate to the ability of the cystic follicular fluids to induce ooeyte maturation, and ooeytes from cystic follicles possess the ability to form male pronncleus even though most were abnormal after in vitro fertilization. 1998 by ElsevierScience Inc.

Key words: bovine, cystic follicle, oocyte, IVF Acknowledgement This study was supported in parts by a grant-in-aid for the Encouragement of Young Scientists (No. 08760291) from the Ministry of Education, Science, Sports and Culture, Japan. a Correspondence and reprint request Theriogenology 50:307-320, 1998 © 1998 by Elsevier Science Inc.

0093-691X/98/$19.00 PII S0093-691X(98)00139-3

Theriogenology

308 INTRODUCTION

Follicular cysts are generally defined as anovulatory follicular structures that are at least 2.5 cm in diameter and that persist for more than 10 d in the absence of a CL (I 1,17). Previous studies suggested that nutritional plane, time after parturition, season of the year, milk production and heritability may be possible factors predisposing to the degeneration of cystic follicles. In more recent studies, it was suggested that an endocrine imbalance involving the hypothalamohypophyseal-gonadal axis is the primary cause of follicular cysts (17). Hamilton et al. (14) speculated that the lack of a strong negative feedback mechanism of estradiol-171~ is the cause of cystic ovarian degeneration. In ruminants, an' average of 40 d is required for early antral follicles to reach the preovulatory stage. The first phase of follicular development up to 3 to 4 mm is not strictly dependent on gonadotropins, in contrast to the final phase of development up to the preovulatory stage (22). Thus, it is essential to include the late phase of follicle development in any study of cystic follicular degeneration, in general, oocytes in healthy follicles are maintained in meiotic arrest until atresia occurs or the LH surge provides a signal for the resumption of meiosis leading to ovulation (16,19). Recently, two hypotheses on resumption of meiosis have been proposed. In one it occurs due to the decreased effects of meiotic inhibitors, mediated by a reduction in the number of gap junctions between granulosa cells. In the other hypothesis meiotic arrest is overcome through stimulation generated within the follicular milieu (28,29). Richard and Sirard (26,27) reported that in vitro cultured theca cells secrete a meiotic arresting factor which is soluble in the medium and acts through the cumulus cells. On the other hand, other studies have shown that addition of follicular fluid to the maturation medium promotes in vitro maturation of bovine (6,18,20,29) and pig (8,24,25,32,33,34) oocytes. Carolan et al. (6) studied the effect of follicular fluid on in vitro maturation of bovine oocytes and found that it is not affected by the size but by the quality of the follicle. From this result, they suggested that the signal conferring atresia on the oocytes is not contained within the follicular fluid. Romero and Seidel (28,29) also reported that exposure of oocytes to follicular fluid collected at various times after the LH surge profoundly affects the rate of cumulus expansion, cleavage and blastocyst formation. Therefore, it was suggested that follicular fluid provides a suitable environment for in vitro oocyte maturation, including cytoplasmic development. Since cystic follicular degeneration seems to be related to meiotic arrest or lack of resumption of meiosis, investigation of the maturation, fertilization and developmental characteristics of oocytes in cystic follicular fluid used, as whole maturation medium, may be helpful to understand the characteristics of the cystic follicle. We wished 1) to investigate the ability of bovine follicular fluid from cystic follicles to promote in vitro maturation and fertilization of oocytes derived from small follicles, 2) to determine progesterone (P4) and estradiol-1713 (E2) concentrations in each cystic follicular fluid and 3) to examine the capability of oocytes derived from homologous cystic follicles to mature and be fertilized in vitro. MATERIALS AND METHODS Experiment I. Comparison of follicular fluid from cystic and normal follicles. Collection of follicular fluid. We compared 3 kinds of follicular fluid: I) Fluid from spontaneously occurring cystic follicles > 30 mm and present for at least 10 d in Holstein Fdesian cows (n=5). The anamnesis for the cows is shown in Table I. Presence of the follicles was diagnosed by palpation per rectum and ultrasonography, the cystic follicles located by ultrasonography and aspirated through the paralumbar fossa with a 23 cm x 1.2 mm needle

a

5,000-10,000 IU, b 200 ~g

Cow No. Interval from detection to follicle aspiration (days) I 20 2 11 3 17 4 39 5 35 hCGa, GnRHb None hCG hCG GnRH

Treatment before aspiration

Interval from treatment to aspiration (days) I (hCG), 20 (GnRH) None 10 27 16

Interval from aspiration to first ovulation 14 None 25 42 26

Table I. The clinical history of cows diagnosed with follicular cysts in Experiment 1.

Pregnant Cull Pregnant Pregnant Pregnant

Exitus after aspiration

C.O 0

O

2

310

Thefiogenology

attached to a 20 ml syringe. 2) Fluid aspirated from small (1 to 7 mm) and large (10 to 20 mm) follicles on normal ovaries collected from Holstein Friesian cows within 20 min after death at a slaughterhouse; these fluids were pooled by size of follicles. 3) Fluid from cystic follicles > 30 mm in diameter on ovaries without a functional CL collected from Holstein Friesian cows (n=5) within 20 min after death at a slaughterhouse. Three cows had multiple cystic follicles, thus a total of 8 cystic follicular fluid samples were collected. All fluids were aspirated by means of a 20 ml syringe and transported to the laboratory in a chilled physiological saline solution. At the laboratory all collected fluids were centrifuged at 1000xg at 4°C for 30 min and stored at -80"C. Follicular fluids were not pooled but investigated separately. Collection and maturation of follicular oocytes. Ovaries collected from Holstein heifers and cows at a local slaughterhouse were transported to the laboratory in saline maintained at 30 to 35°C. Cumulus-oocyte complexes were collected by puncturing follicles 1 to 7 mm in diameter using an 18-gauge needle attached to a 5 ml plastic syringe containing 0.5 to 1.0 ml of modified phosphate buffered saline (PBS; Embryotec: Nihon Zenyaku Co., Fukushima, Japan) supplemented with 100 IU/ml penicillin and 100mg/ml streptomycin (Meiji Seika Co. Tokyo, Japan). Collected COCs were then washed twice with PBS and immediately used for in vitro culture. The COCs (n=10-15) were transferred to a 100 pl droplet of one of 3 maturation media consisting of 100% cystic follicle fluid from live cows, and fluid from small and large non-cystic follicles collected post mortem in Petri dishes (Falcon 1008) and cultured for 24 h at 38.5°C in 5% CO2 in air under paraffin oil (Sigma). All the above media were supplemented with I IU PMSG, 100 IU/ml penicillin and 100 pg/ml streptomycin and 2 mM sodium pyruvate. Additionally, TCMI99 with Earle's salts (Gibco BRL, Life Technologies Inc., Grand Island, NY, USA) supplemented with 5% (v/v) superovulated cow serum (SCS, 4), with (+) or without (-) PMSG served as a control medium. After 24 h of in vitro maturation, COC's were observed under a stereomicroscope and the degree of cumulus expansien recorded according to Eppig et al. (10). ,In vitro fertilization of in vitro matured oocytes. Frozen semen samples (0.5 ml straw) from 3 Holstein bulls were thawed in a 38°C water bath, and pooled. An aliquot of the thawed semen mixture was placed in to conical tubes (Falcon 2054) under 1 ml of Brackett and Oliphant (BO) solution supplemented with 2.5 mM caffeine (Sodium benzoate e4144, Sigma). After incubation for I h at 38.5°C, the top 0.8 ml was collected. The spermatozoa were washed twice (500xg, 5min) with capacitation medium and resuspended at a density of I to 2 x 106 cell/nil in 100 pl of a 50:50 mixture of BSA-free BO solution with 2.5 mM caffeine and BO solution with 20 mg/ml BSA (Sigma, USA) and 10 pg/ml heparin (Shimizu Pharmaceutical Co., Ltd., Shimizu, Japan). In vitro matured COCs were washed 3 times with a BO solution containing 10 mg/ml BSA, and 10 to 15 COCs were transferred to a 100 pl sperm suspension drop under paraffin oil and co-incubated for 18 h at 38.5 °C in 5% CO2 in air. Assessment of sperm penetration, male pronucleus formation and polyspermy. After 18 h of cultu~re for in vitro fertilization, the cumulus cells of the COCs were removed by pipetting and whole-mount preparations of oocytes were fixed for 3 days with an ethanol:acetic acid mixture (3: !, v/v), stained with 1 % aceto-orcein and examined under a stereomicroscope (x400). The presence of unswollen or swollen sperm head(s) or male pronuclei in the ooplasm was taken as evidence for sperm penetration. The presence of two or more pronuclei with a second polar body in the perivitelline space or with a detached sperm midpieee in the ooplasm was taken as evidence of male pronucleus development. The presence of multiple male pronuclei in cytoplasm was considered indicative of polyspermy. This experiment was replicated each 3 times using the 3 kinds of follicular fluids described above.

Theriogenology

311

Enzyme immunoassay of steroid hormones in follicular fluid. Steroid hormone concentrations in cystic follicular fluid were measured using double antibody immunoassays (EIAs) in 96 well ELISA plates (Coming, New York, U.S.A.) coated with 50/~l of antirabbit lgG (Seikagaku Co. Tokyo, Japan). The progesterone (P4) EIA was performed as described by Miyamoto et al. (21). The estradiol-1713 (E2) EIA was based on P4 EIA with minor modifications. Basically, 30 ,ul of standard and samples was incubated with 100/~l (l:100,000) polyclonal antibody for 25 hrs at 30°C, decanted, supplemented with 100 /~l (i:8,000) E2-6CMOhorseradish peroxidase (E2-HRP) and incubated for a further 2 hrs at 30°C. The sensitivity was 3.9 pg/ml for E2 and 0.05 ng/ml for P4. The intra-and inter-assay coefficient of variations were 3.8% and 6.4% for E2, and 5.3% and 8.1% for P4, respectively. From the 1:'4 and E2 concentrations, the P4/E2 ratio was calculated and the cystic follicular fluids were divided into two groups as P4/E2I. Experiment 2: Evaluation of the ability of I) COC's sampled from cystic follicles to form the pronucleus after in vitro maturation / fertilization, and 2) follicular fluids sampled from the homogenous cystic follicles to promote in vitro maturation/fertilization of COC's from small follicles. .Sample collection from a slaughterhouse. Ten cystic ovaries were collected at a slaughterhouse as described in Experiment 1. The COCs from the cystic follicles were collected as in a previous report (20). Briefly, intact cystic follicles were dissected free from the ovarian cortex, washed well with physiological saline, ruptured into 90 mm petri dishes, and the O3Cs searched under a microscope. Collected COCs were classified into 5 groups according to Goto et al. (13), based on the morphology of their surrounding cumulus cell i.e : many layers of compact cumulus cells (Group A); partially removed cumulus cells (Group B); denuded oocytes (Group C); degenerated oocyte cytoplasm (Group D) and expanded cumulus cells (Group E). Follicular fluid from the cystic follicles corresponding to those oocytes classified between A and E were used for in vitro maturation / fertilization of oocytes from small follicles. The fluids from the ruptured cystic follicles were immediately collected and the volume measured. Fluid from cystic follicles containing more than l0 ml was centrifuged at 4°C and the supematant stored as in Experiment I Ln vitro maturation and fertilization of oocytes from cystic follicles. The C(X~s from cystic follicles were washed with m-PBS, individually transferred to a 100 pl droplet of TCM199 with Earle's salts supplemented with 5% SCS, 1001U/ml penicillin and 100 pg/ml streptomycin and 2 mM sodium pyruvate with 1 IU PMSG, and cultured for 24 h at 38.50C in 5% CO2 in air. The COCs from small follicles matured individually under the same conditions served as controls. After in vitro maturation, the COCs were washed 3 times with a BO solution containing 10 mg/rnl BSA and in vitro fertilization was conducted as described in Experimant 1. In vitro maturation of the COCs from small follicles with 100% each cystic follicle fluids, subsequent in vitro fertilization, assessment of the oocytes after in vitro fertilization and the steroid hormone assay, were all conducted as in Experiment I. Statistical analysis The significance of the effect of cystic follicular fluids as maturation medium on subsequent sperm penetration, male pronucleus formation and polyspermy compared with control groups was analyzed using analysis of variance (ANOVA) with Stat View 4.0 computer program (1). Differences were considered to be significant at P< 0.05.

312

Theriogenology RESULTS

Experiment 1 The degree of cumulus expansion, sperm penetration rate, male pronucleus formation rate, polyspermy rate and concentration of P4 and E2 in fluid from cystic follicles collected from live cows are shown in Table 2. The morphological profile of cumulus expansion of COCs matured for 24 h in TCMI99 based maturation medium and follicular fluid collected from cystic follicles are presented in Figure I. Cumulus expansion clearly differed between TCMI99 (+) and TCMI99 (-). The cumulus expansion of the COCs matured with fluid from cystic follicles collected from live cows was greater than that of TCMI99 (+) except in L-CFF-2 and L-CFF-3 where the expansion was not induced. There were no significant differences in sperm penetration (87.0-94.6%), male pronucleus formation (77.6-86.8%) and polyspermy (16.8-25.4%) among control groups (TCMI99, Pooled follicular fluids from small follicles and large follicles). However, the sperm penetration (56.5%) and male pronucleus formation (49.3%) in L-CFF-I were significantly low when compared to control groups. Also, the sperm penetration rate of LCFF-3 was significantly low (76.0%) relative to the pooled fluid from small and large follicles. The P4 and E2 concentrations and P4~2 ratio in 5 individual cystic follicular fluids from live cows ranged from < 0.25 to 268.2, 0.07 to 0.41 ng/ml and < 3.6 to 2980, respectively. The effect of cystic follicular fluid from slaughtered cows on the cumulus expansion, sperm penetration, male pronucleus formation and polyspermy in bovine oocytes and the relationship to the steroid hormone concentration in the cystic follicular fluid are shown in Table 3. The cumulus expansion of the COCs matured with cystic follicular fluids was greater than that of TCMI99 (+). There were no significant differences in sperm penetration (83.2-95.8%), male pronucleus formation (76.8-89.0%) and polyspermy (12.6-33.6%) among control groups (TCMI99, Pooled follicular fluids from small and large follicles) except for male pronucleus formation (62.8%) of CFF-2. There were no significant differences in sperm penetration, male pronucleus formation and polyspermy between P4/E2>I and P4/E2
65

63

54

L-CFF-3

L-CFF-4

L-CFF-5

+++

+++

+++

+++

+++

+

++

47 (86.0----. 15.0)

56 (86.3± 17.5)

51 (76.0+15.6) bc

80 (90.8 "1- 2.6)

34(56.5 -t- 8.9 )b

6 0 ( 9 1 . 5 ± 9.5) a

55 (94.6 -t- 5.6) a

150 (87.0+ 11.6) ac

141 (88.9 -I- 7.2) ac

Sperm penetration

42 (75.6-----33.0)

48 (72.8"+'23.2)

49(72.7+15.4)

76 (86.0 .+. 5.2)

30 (49.3 -t- 12.2) b

50 (77.6+15.1) a

50 (85.4----- 8.8) a

142(81.8"+'12.6) a

138(86.8 -I- 8.1) a

Male pronucleus formation

No. (%----.SD) of oocytes

11 ( 18.6 -I- 19.0)

13 (20.2"1"25.9)

II (18.6 + 9.9)

24 (26.8± ! l.O)

5 ( 9.1 + 12.7)

15(25.4+11.9)

12 (21.2 -+- 3.3)

41 (23.2"+'12.5)

26(16.8 "1- 5.8)

Polyspermy

TCM(+) with PMSG, TCM(-) without PMSG, SFF Pool of follicular fluids from small follicles (l-7mm) LFF Pool of follicular fluids from large follicles (lO-20mm) a-c Values within a column with different superscripts are significantly different (P
58

88

65

LFF

L-CFF-2

59

SFF

L-CFF-1

158

172

TCMI99 (-)

No.of Cumulus oocytes expansion

TCMi99(+)

Media

153.2

i.8

157.7

< 0.25

268.2

26.4

35.1

P4 (ng/ml)

0.10

0.41

O. l l

0.07

0.09

16.8

4.3

E2 (ng/ml)

Concentration

Table 2. Effects of maturation media and bovine cystic and normal follicular fluids from live cows (L-CFF) on cumulus cell expansion, penetration, pronucleus formation and polyspermy in bovine oocytes and the concentrations of progesterone (P4) and estradiol-1713 (E2).

1531.8

4.3

1433.2

< 3.6

2980.0

1.6

8.2

P4/E2

f.o (Jo

t~

314

Theriogenology

Figure 1. (A) Cumulus-oocytes complexes matured in TCM 199 with PMSG for 24 h showing a slight degree of expansion of all the layer of cumulus cells. (B) matured in TCM 199 without PMSG for 24 h showing no expansion of the cumulus cells, (C) matured in cystic follicle fluid from live cow-I for 24 h showing greater degree of expansion of all the layers of the cumulus cells than TCM 199 with PMSG, (D) matured in cystic follicle fluid from live cow-3 for 24 h showing no expansion of the cumulus oophorus.

50

49

64

76

CFF2

CFF 3

CFF 4

CFF 5

51

56

CFF7

CFF 8

+++

+++

+++

+++

+++

+++

+++

+++

Cumulus expansion

47 (83.24-20.8)

49(94.44- 5.1)

50(94.14- 5.8)

73 (95.84- 1.2)

55 (87.34- 7.6)

47 (95.3 4- 5.0)

43(86.1 + 6.7)

56 (88.0 + 12,5)

Sperm penetration

44 (77.1 4-20.7)

42 (80.64- 7.6)

45 (83.8 4- 8.4)

69 (89.0+ 9.2)

54 (86.2 .4- 9.5)

43 (87.2+ 6.3)

32(62.8+11.1) a

49 (76.8 + 13.8)

17 (33.64"30.5)

8 (19.4+18.0)

8 (12.6 + 9.2)

20 (27.84- 6.8)

17 (26.5 + 9.4)

12 (30.84-25.5)

9(17.8 + 3.8)

17 (28.3+ 8.6)

No.(% 4- SD) of oocytes Male pronucleus Polyspermy formation

8.7

13.9

23.9

121.2

128.1

7.3

289.4

128.8

9. !

19.4

51.5

0.01

0.01

5.6

0.04

0.14

Concentration P4 F,2 (ng/ml) (ng/ml)

P4 Progesterone, E2 Estradiol-171$ a Significantly different (P<0.05) between male pronucleus formation rate of TCM 199(+) in Table 2.

54

CFF6

P4/E2< 1

63

Pa/E2> 1

CFF l

No.of oocytes

Table 3. The relationship between the effect of bovine follicular fluids from cystic follicles (CFF) on penetration, male pronucleus formation and polyspermy in bovine oocytes and the steroid hormone concentration in the CFF.

0.95

0.72

0.46

12118

12808

1.3

7235.3

920.1

P4/E2

O1

ca

A A A C D D D E

A C

IP+3E 6P+IE 2P+2E MII 2P+5E 3P+IE 3P+5E Degeneration

2P 1P+ 1E

Pronucleus formation after IVF

93.2 (44/47) 79.5 (33/44) 96.1 (49/51) 90.4 (47/52) 96.4 (55/57) 87.6 (50/57) 90.4 (54/60) 81.2 (47/58) 92.5 (71/77)

94.2 (50/53)b 94.7 (51/54)

Male pronucleus formation (%)

12.5 31.3 28.1 39.1 26.8 24.4 44.9 21.0

41.6 123.2

36.5 62.0 126.0 106.6 72.2 96.3 63.8 139.7

38.5 4.3

Concentration P4(ng/ml) E2(ng/ml)

Pa Progesterone, E2 Estradiol-1715, P Pronucleus, E Enlarged sperm head a In vitro maturation with TCM-199 based maturation medium, b Oocytes which form male pronucleus / total no.of oocytes

P4/E2< 1 3 4 5 6 7 8 9 I0 Controla

P4/E2> I 1 2

Follicle Rank of No. COCs

0.34 0.5 0.22 0.37 0.37 0.25 0.7 0.15

1.1 28.9

P4/E2

Table 4. The morphology of the cumulus-oocytes complexes, the profile of the male pronucleus formation after in vitro fertilization and the steriod hormone concentration of the follicular fluid from cystic follicles.

GO ....t

Theriogenology

317

DISCUSSION The results of Experiment I indicate that bovine cystic follicular fluids supplemented with PMSG possess the ability to induce cumulus expansion, nuclear maturation of bovine oocytes, and the fertilization, similar to those in pooled follicular fluid from small and large follicles and TCM 199 based culture media. Moreover, excellent cumulus expansion was observed in all cystic follicular fluids except for L-CFF-2 and 3. In L-CFF-2 and 3, the male pronucleus formation was not different from control groups. In the case of L-CFF-I, although the cumulus expansion was greater than TCMI99 (+), sperm penetration and male pronucleus formation were significantly inhibited. Sirard (31) suggested that cumulus expansion is not necessary to achieve normal nuclear configuration during in vitro maturation. Daen et al. (9) also reported that increased cumulus expansion does not correlate directly with the enhancement of male pronucleus formation and suggested that the active factor(s) involved in cumulus expansion and male pronucleus formation are not the same. The results of the present study reconfirmed the previous findings and suggest that follicular fluid from cystic follicle also possess the factors responsible for cumulus expansion and oocytes maturation. However, Romero and Seidel (29) reported a positive relationship between in vitro cumtflus expansion and the rate of cleavage and blastocyst formation. Further investigations are needed to clarify the effect of follicular fluid from cystic follicle on oocyte maturation, especially on cytoplasmic maturation. Sirard (30) suggested that bovine COCs cultured for 24 h in whole fluid from small follicles reduced oocyte maturation and inhibited nucleus maturation. Moreover, Ayoub and Hunter (2) reported that the bovine follicular fluid from small (2 to 4 mm), medium (5 to 9 mm) and large (10 to 20 mm) follicles inhibited the resumption of meiosis. However, in the present study, sperm penetration and male pronucleus formation in the follicular fluid from small and large follicles were similar to TCM 199 groups as controls. We did not evaluate the maturation rates, and the reason for this conflicting result is unclear, though it might be due to differences in culture conditions. The concentrations of P4 and E2 in follicular fluids didn't show any relationship with cumulus expansion, sperm penetration, male pronucleus formation or polyspermy except in LCFF-I. Moor et al. (23) reported that alterations to the steroid profile during maturation of the follicles induced changes in the oocyte which were expressed as gross abnormalities at fertilization. However, in mammals the role of steroids in oocytes maturation is still controversial (3) and needs further investigations. Cook et al. (7) reported three phenomena that occur in cows with cysts induced by steroid treatment; turnover, persistence, and spontaneous recovery of ovarian cyclicity. Hamilton et al. (14) reconfirmed the above and additionally reported that the mean interval from the initial detection of a follicle/cyst wave to detection of a new follicle/cyst wave in cows was relatively long and variable (6 to 26 days). In the case of multiple cystic follicles in the ovaries in the present study (CFF3 and CFF6, CFF4 and CFF5, CFF7 and CFF8), the size of the follicles and the hormonal profiles including the P4/E2 ratio of follicular fluid was the same, with the exception of the steroid hormone concentration of CFF3 and CFF6. Thus the ability of the follicular fluids from cystic follicles to induce oocyte maturation appeared to remain unchanged regardless of the age of the follicles. The results of Experiment 2 suggest that the morphology of the COCs from cystic follicles is not related to the ability of follicular fluids from cystic follicles to induce oocyte maturation or

318

Theriogenology

P4 and E2 concentrations. Further, these oocytes from cystic follicles possess the ability to form male pronucleus even though polyspermy was observed after in vitro fertilization. The concentration of P4 and E2 was lower and higher respectively, in follicular fluids from cystic follicles of experiment 2 than cystic follicular fluids collected from live cows of Experiment I. Hernandez-Ledezma et al. (15) reported that mean concentrations of E2 in the follicular fluids from ovarian cyst and normal follicles did not differ but were highly variable. Further, GnRH treatment of cows with ovarian cysts modifies the steroidogenic ability of the cysts and induced LH release which appears to stimulate luteinization of the cyst and increase the P4 in follicular fluids. The high levels of P4 in fluids from cystic follicles collected from live cows observed in experiment I may be due to the GnRH or hCG treatments prior to aspiration of the follicular fluids. Fukui et al. (12) reported that of the oocytes collected from the follicles, 40.3% were degenerated, and 28.6 and 15.6% were at the germinal vesicle and Metaphase II stage of meiotic division, respectively. So, although the stage of the meiotic division of the oocytes from cystic follicles before in vitro maturation / fertilization is unknown in the present study, the results suggested that the cytoplasm, especially the function of cortical granules, is abnormal, even in those oocytes at the germinal vesicle stage. In general, the steroid hormone content of follicular fluids reflects the synthetic capacity of theca cells (5). Therefore, the E2 concentrations of the E2-dominant cystic follicular fluids in this study might reflect the synthetic capacity and viability of the theca cell layers. On the other hands, Richard and Sirard (26,27) recently showed that theca cells secrete factor(s) for the maintenance of meiotic arrest. Our results as to the ability of the follicular fluids from cystic follicles to induce male pronucleus formation after in vitro maturation / fertilization, suggest that the synthetic capacity of steroid hormones of theca cells does not reflect the capacity for secretion of meiotic arresting factors. In conclusion, many of the bovine follicular fluids from cystic follicles supplemented with PMSG possess the ability to induce cumulus expansion, nuclear maturation of bovine oocytes and male pronucleus formation following in vitro maturation / fertilization, similar to follicular fluids from normal small and large follicles. The morphology of the cumulus-oocytes complexes from cystic follicles might not relate to the ability of the follicular fluids from cystic follicles to induce in vitro maturation of oocytes from small follicles, and the oocytes from cystic follicles possess the ability to form male pronucleus formation even though most were abnormal after in vitro maturation / fertilization. REFERENCES 1. Abacus concepts-Stat-View 4.0 Abacus concepts, Inc., Berkeley, California, 1992. 2. Ayoub MA and Hunter AG. Inhibitory effect of bovine follicular fluid on in vitro maturation of bovine oocytes. J Dairy Sci 1993; 76: 95-100. 3. Bevers MM, Dieleman SJ, Van den Hurk R and lzadyar F. Regulation and modulation of oocyte maturation in the bovine. Theriogenology 1997; 47: 13-22. 4. Boediono A, Takagi M, Saha S and Suzuki T. Influence of Day-0 and Day-7 superovulated cow serum during development of bovine oocytes in vitro. Reprod Fertil Dev 1994; 6: 261-264. 5. Borromeo V, Bramani S, Berrini A, Sironi G, Finazzi M, Cremonesi F and Secchi C. Growth hormone but not prolactin concentrations in the fluid of bovine ovarian cysts are related to the cystic stage of luteinization. Theriogenology 1996; 46: 481-489.

Theriogenology

319

6. Carolan C, Lonergan P, Monget P, Monniaux D and Mermillod P. Effect of follicle size and quality on the ability of follicular fluid to support cytoplasmic maturation of bovine oocytes. Mol Reprod Dev 1996; 43: 477-483. 7. Cook DL, Smith CA, Parfet JR, Youngquist RS, Brown EM and Garverick HA. Fate and turnover rate of ovarian follicular cysts in dairy cattle. J Reprod Fert 1990; 90: 37-46. 8. Daen FP, Sato E, Naito K and Toyoda Y. The effect of pig follicular fluid fractions on cumulus expansion and male pronucleus formation in porcine oocytes matured and fertilized in vitro. J Reprod Fertil 1994; 101: 667-673. 9. Daen FP, Sato E, Nakayama T and Toyoda Y. Serum factor(s) stimulating cumulus expansion in porcine oocyte-cumulus complexes matured and fertilized in vitro. Cell Struct Funct 1995; 20: 223-231. 10. Eppig J J, Wigglesworth K and Chesnel F. Secretion of cumulus expansion enabling factor by mouse oocytes: Relationship to oocyte growth and competence to resume meiosis. Dev Biol 1993; 158: 400.409. 1 I. Eyestone WH and Ax RL. A review of ovarian follicular cysts in cows, with comparisons to the condition in women, rats and rabbits. Theriogenology 1984; 22: 109-125. 12. Fukui Y, ishiguro A, Fukushima M, Terawaki Y and Ono H. Plasminogen and other chemical components in the fluid of bovine cystic and normal-like follicles. Res Bull Obihiro Univ 1983; 13:167-173 (in Japanese). 13. Goto K, Tanimoto Y, Fujii W, Taniguchi S, Takeshita K, Yanagita K, Ookutsu S and Nakanishi Y. Evaluation of once-versus twice-weekly transvaginal ultrasound-guided follicular oocyte aspiration with or without FSH stimulation from the same cows. J Reprod Dev 1995; 41: 303-309. 14. Hamilton SA, Garverick HA, Keisler DH, Xu ZZ, Loos K, Youngquist RS and Salfen BE. Characterization of ovarian follicular cysts and associated endocrine profiles in dairy cows. Biol Reprod 1995; 53: 890-898. 15. Hernandez-Ledezma J J, Garverick HA, Elmore RG, Brown EM and Kesler DJ. Gonadotropin releasing hormone treatment of dairy Cows with ovarian cysts. Iil. Steroids in ovarian follicular fluid and ovarian cyst fluid. Theriogenology 1982; 17: 697-707. 16. Hyttel P, Callesen H and Greve T. Ultrastructure features of preovulatory oocyte maturation in superovulated cattle. J Reprod Fertil 1986; 76: 645-656. 17. Kesler DJ and Garverick HA. Ovarian cysts in dairy cattle: a review. J Anim Sci 1982; 55: 1147-1159. 18. Kim KS, Mitsumizo N, Fujita K and Utsumi K. The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos. Theriogenology 1996; 45: 787-799. 19. Kruip TAM, Cran DG, Van Beneden TH and Dieleman SJ. Structural changes in bovine oocytes during final maturation in vivo. Gamete Res 1983; 8: 29-47. 20. Lonergan P, Monaghan P, Rizos D, Boland MP and Gordon I. Effect of follicular size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro. Mol Reprod Dev 1994; 37: 48-53. 21. Miyamoto A, Okuda K, Schweigert FJ and Schams D. Effects of basic fibroblast growth factor-13 and nerve growth factor on the secretory function at the bovine corpus luteum in vitro. J Endocrinology 1992; 135:103-114. 22. Monniaux D, Monget P, Besnard N, Huet C and Pisselet, C. Growth factors and antral follicular development in domestic ruminants. Theriogenology 1997; 47: 3-12. 23. Moor R.M, Polge C and Willadsen S M Effect of follicular steroids on the maturation and fertilization of mammalian oocytes. J Embryol Exp Morph 1980; 56: 319-335. 24. Naito K, Fukuda Y and Toyoda Y. Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro. Gamete Res 1988; 21: 289-295.

320

Theriogenology

25. Naito K, Kosaka M, Fukuda Y, Ishibashi I and Toyoda Y. Analysis of the factor(s) present in follicular fluids promoting male pronucleus formation ability of porcine follicular oocytes. Jpn J Anim Reprod 1990: 36: 213-218. 26. Richard FJ and Sirard MA. Effects of follicular cells on oocyte maturation. I: Effects of follicular hemisections on bovine oocyte maturation in vitro. Biol Reprod 1996a; 54: 16-21. 27. Richard FJ and Sirard MA. Effects of follicular cells on oocyte maturation. I1: Theca cell inhibition of bovine oocyte maturation in vitro. Biol Reprod 1996b; 54: 22-28. 28. Romero-Arredondo A and Seidel GE Jr. Effects of bovine follicular fluid on maturation of bovine oocytes. Theriogenology 1994; 41: 383-394. 29. Romero-Arredondo A and Seidel GE Jr. Effects of follicular fluid during in vitro maturation of bovine oocytes on in vitro fertilization and early embryonic development. Biol Reprod 1996; 55: 1012-1016. 30. Sirard MA and First NL. In vitro inhibition of oocyte nuclear maturation in the bovine. Biol Reprod 1988; 39: 229-234. 3 I. Sirard MA. Temporary inhibition of meiosis resumption in vitro by adenylate cyclase stimulation in immature bovine oocytes. Theriogenology 1990; 33:757 -767. 32. Sluss PM, Schneyer AL, Franke MA and Reichert LE Jr. Porcine follicular fluid contains both follicle-stimulating hormone agonist and antagonist activities. Endocrinology 1987; 120: 1477-1481. 33. Yoshida M, lshigaki K and Pursel V. Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro. Mol Reprod Dev 1992; 31:68-7 I. 34. Yoshida M, Ishizaki Y, Kawagishi H, Bamba K and Kojima Y. Effects of pig follicular fluid on maturation of pig oocytes in vitro and on *,heirsubsequent fertilizing and developmental capacity in vitro. J Reprod Fertil 1992; 95: 481-488.