Effect of protein synthesis inhibition before or during in vitro maturation on subsequent development of bovine oocytes

EI~EVIER

EFFECT OF PROTEIN SYNTHESIS INHIBITION BEFORE OR DURING IN VITRO MATURATION ON SUBSEQUENT DEVELOPMENT OF BOVINE OOCYTES P. Lonergan, 1 T. Fair, H. Khatir, G. Cesaroni and P. Mermillod INKA-PRMD, 37380 Nouzilly, France Received for publication: December 15, 1997 Accepted:May 20, 1998 ABSTRACT The overall objective of this study was to assess the effect of maintaining meiotic arrest in bovine oocytes in vitro on developmental competence. In Experiment 1 the effect of inhibition of meiotic resumption using cyeloheximide (CX),on subsequent was examined. Immature cumulus ooeyte complexes (COCs, n=804) were cultured in the absence (24 h) or presence of CX for 6, 12, 18 or 24 h. The control was inseminated 24 h later, while CXtreated ooeytes were cultured for a further 24 h before insemination. In Experiment 2 the effect of exposing the ooeyte (n=1239) during meiotic arrest to putative stimulatory substances (pFSH and FCS) was examined. In Experiment 3, to study the importance of protein synthesis during maturation, synthesis was blocked for a 6-h period at various times (6, 12, 18 h) after start of culture (n=ll17). In Experiment 1, there was no difference in cleavage rate between treatments. However, the percentage of 5 to 8 cell embryos at 72 h post insemination was significantly lower after CX treatment (64 vs 42 to 51%; P<0.05). This was reflected in a lower rate of blastoeysts at Day 6 (9 to 15 vs 31%, P<0.002). While the blastoeyst rate at Day 8 was lower in CX-treated oocytes, the effect was only significant when CX was present for longer than 12 h. A marked decrease in development was noted following inhibition for 18 h or more compared with the control (17 to 19 vs 40%; P<0.0002). In Experiment 2, addition of either FSH or FCS to oocytes in the presence of CX had no effect on any of the parameters studied, even though there was a positive effect in control oocytes. In Experiment 3, treatment with CX after the oocytes had matured for varying periods resulted in decreased blastocyst rates at Days 6 and 8 of culture. The most significant drop in development occurred when oocytes were cultured for 12 h before exposure to CX (15 vs 40%; P<0.0001). In conclusion, CX-blocked ooeytes retained their developmental competence, although final blastoeyst yields were reduced. © lg98 by ElsevierScience Inc.

Key words: bovine, oocyte, meiotic arrest, eycloheximide, protein synthesis Acknowledgements P. Lonergan was a recipient of a grant from the Foundation pour la Recherche Medicale, France. 1Correspondence and present address: Department of Animal Science and Production, University College Dublin, Lyons Research Farm, Newcastle, County Dublin, Ireland. Fax: +353 1 6288421, E-mail: [email protected] Theriogenology 50:417-431, 1998 © 1998 by Elsevier Science Inc,

0093-691)(/98/$19.00 PII S0093-691X(98)00149-6

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INTRODUCTION During fetal life, mammalian oocytes initiate meiosis and become arrested at the diplotene stage of prophase I, the so-called dictyate stage. The ability of these oocytes to resume meiosis and to complete the first meiotic division is acquired sequentially during their growth phase (8, 27). In fully grown oocytes, meiotic resumption and nuclear maturation, in response to the preovulatory gonadotropin surge in vivo or release from the follicle in vitro, is characterized by germinal vesicle breakdown (GVBD), chromosomal condensation, progression through metaphase I to anaphase and telophase, with extrusion of the first polar body and arrest at metaphase II (MII) until reactivation at fertilization. Because such a high proportion (>90%) of bovine oocytes achieves nuclear maturation to MII under routine culture conditions, researchers in bovine IVF have concentrated on the process of cytoplasmic maturation, measured as the ability of the oocyte to develop after fertilization (16, 22, 23). In most laboratories, blastocyst production in vitro reaches a plateau at 30 to 40% of inseminated oocytes, in spite of numerous variations on the basic technique. It has become clear that germinal vesicle (GV) stage oocytes, or indeed ooeytes at MII, are not developmentally equivalent to each other. The poor development observed in vitro is thought to be due to the intrinsic quality of the oocyte itself rather than to suboptimal culture conditions, although a role for the latter is certainly not excluded (see reference 7). To understand the mechanisms responsible for the acquisition of developmental competence, it is necessary that the latter stages of follicular development be simulated in vitro. A prerequisite for this is the establishment of a culture system that reproduces the ovarian follicular environment for the oocyte, in which meiotic resumption and nuclear maturation are prevented. We have previously shown that it is possible to reversibly inhibit meiotic resumption in bovine oocytes for 24 h using CX, an inhibitor of protein synthesis, with over 80% of blocked oocytes reaching metaphase II after removal of the inhibitory conditions and about 20% developing to the blastocyst stage (23). Similar findings have subsequently been reported by Saeki et al. (36). While encouraging, the developmental rates following artificial maintenance of meiotic arrest by protein synthesis inhibition are still inferior to those obtained with non arrested oocytes. The importance of protein synthesis in oocyte meiotic resumption has been clearly shown in several mammalian species, including cattle (11, 13, 14, 37), pigs (9, 18), sheep (26) and goats (19). In immature bovine oocytes new protein synthesis during the first 8 h of culture is indispensable for meiotic resumption (14, 42). This is in contrast to certain rodent oocytes, in which protein synthesis is not required for GVBD (28, 31, 41). The aim of the work presented here was 1) to examine the effect of inhibition of meiotic resumption/GV breakdown (GVBD) for varying periods after initiation of culture (6, 12, 18, 24 h) by protein synthesis inhibition on subsequent oocyte development to identify the point at which development becomes compromised, following on from our previous work in which 24 h of inhibition was shown to be detrimental; 2) to assess the effects of stimulating the oocyte with putative growth-promoting substances (FCS, FSH) during the period of CXinduced arrest in order to improve cytoplasmic maturation and thus subsequent development; and 3) to determine the importance of protein synthesis during maturation by inhibiting

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synthesis for a short period (6 h) at various times during the process of maturation and observing its effect on development. MATERIALS AND METHODS Oocyte Recovery, Maintenance of Meiotic Arrest and Subsequent Maturation In Vitro Chemicals were purchased from Sigma Chemical Co (St. Louis, MO, USA) unless otherwise indicated. A stock solution of 10 mg/mL CX was prepared in Medium 199 (M199), aliquoted and stored at -20°C until use. The pFSH was provided by Dr Yves Combarnous, (INRA, Nouzilly, France). The details of methods for oocyte recovery, in vitro maturation (IVM), fertilization (IVF) and culture (IVC) have been described previously (25). Cumulus-ooeyte-complexes were obtained by aspiration of 2 to 8 mm follicles of ovaries from slaughtered cows. Following 4 washes in modified PBS, supplemented with 36 mg/mL pyruvate, 50 mg/mL gentamycin and 0.5 mg/mL BSA (Sigma, fraction V, cat. # A-9647), groups of up to 50 COCs were transferred to 4-well plates (Nunc, Roskilde, Denmark) contairting 500 p,L of culture medium (see below). Between recovery of the COCs and onset of culture approximately 60 min passed, during which the COCs were left in undiluted follicular fluid. Experiment 1 The objective of this experiment was to examine the effect of artificially maintaining meiotic arrest by inhibition of GVBD using CX for increasing periods after oocyte removal from the ovary on subsequent development. Immature COCs (n=804) were randomly allocated to 1 of the 5 following treatment groups: 1) M199+10% FCS (v/v); 2) M199+10 txg/mL CX for 6 h; 3) M199+CX for 12 h; 4) M199+CX for 18 h; and 5) M199+CX for 24 h. For the control (Group 1), following 24 h of culture, COCs were inseminated as described below. For Groups 2 to 5, following incubation for the specified times in the presence of CX, the COCs were recovered and washed by passing them through 4 dishes of PBS in order to remove residual CX before being cultured for a further 24 h in M199+10% FCS. Thus, all CX-treated groups spent 24 h under conditions conducive to normal maturation (i.e., M199+10% FCS). Following this treatment, oocytes were inseminated and cultured as for the control. Four replicates were carried out, with each treatment group being represented in each replicate. Experiment 2 The objective of this experiment was to examine the effect of exposing the ooeyte during CX-induced meiotic arrest to substances known to stimulate development in untreated oocytes fiSH, FCS) to improve development following removal of inhibitory conditions. 2(a). Cumulus-oocyte-complexes (n=580) were cultured for 24 h in 1) M199 alone or supplemented with 2) 40 ng/mL FSH, 3) 400 ng/mL FSH, 4) 10 ~tg/mL CX, 5) CX+40 ng/mL FSH, or 6) CX+400 ng/mL FSH. After 24 h of culture, Groups 1, 2 and 3 were

420

Theriogenology

submitted to IVF as described below, while groups 4, 5 and 6 were further cultured for 24 h in M199+FCS, following which they were inseminated. Three replicates were carried out, with each treatment group being represented in each replicate. 2Co). Cumulus-oocyte-complexes (n=659) were cultured for 24 h in 1) M199 alone or supplemented with 2) 10% FCS, 3) 10 lag/mL CX, or 4) FCS and CX. After 24 h of culture, Groups 1 and 2 were submitted to IVF, while Groups 3 and 4 were cultured for a further 24 h in M199+FCS. Four replicates were carried out, with each treatment group being represented in each replicate Experiment 3 The objective of this experiment was to examine the effect of inhibition of protein synthesis with CX for a 6-h period at various times during oocyte maturation on subsequent development. Immature COCs (n=l 117) were randomly allocated to 1 of the 6 following treatment groups: 1) M199+10% FCS for 24 h; 2) M199+10 I~g/mL CX for 6 h followed by M199+FCS for 24 h, 3) M199+FCS for 6 h followed by M199+CX for 6 h end M199+FCS for 18 h, 4) M199+FCS for 12 h, MI99+CX for 6 h, M199+FCS for 12 h, 5) M199+FCS for 18 h, M199+CX for 6 h, M199+FCS for 6 h, and 6) M199+FCS for 30 h. Oocytes in Group 1 were inseminated 24 h after initiation of culture, while all other groups were inseminated 30 h after initiation of culture. Thus, all CX-treated groups spent 24 h under conditions conducive to normal maturation (M199+10% FCS). Following incubation in the presence of CX, the COCs were recovered and washed by passing them through 4 dishes of PBS in order to remove residual CX before being further cultured in M199+10% FCS. Following this treatment, oocytes were inseminated and cultured as for the control. Five replicates were carried out, with each treatment group being represented in each replicate. In Vitro Fertilization For IVF, COCs were washed 4 times in PBS and once in fertilization medium before being transferred in groups of up to 50 into 4-well plates containing 250 ~ / w e l l of fertilization medium (TALP, containing 10 Ixg/mL heparin-sodium salt, Calbiochem, San Diego, CA, USA). Motile spermatozoa were obtained by centrifugation of frozen-thawed spermatozoa on a Percoll (Pharmacia, Uppsala, Sweden) discontinuous density gradient (2 mL of 45% Percoll over 2 mL of 90% Percoll) for 20 rain at 700 x g at room temperature. Viable spermatozoa, collected at the bottom of the 90% fraction were washed in TALP and pelleted by centrifugation at 100 x g for 10 min at room temperature. Spermatozoa were counted in a haemocytomcter and diluted in the appropriate volume of TALP to give a concentration of 4 x 106 sperm/mL, then 250 Ixl of this suspension were added to each fertilization well to obtain a final concentration of 2 x 106 sperm/mL. Plates were then incubated for 20 to 24 h in 5% CO2 in humidified air at 39°C.

Theriogenology

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Post-FertilizationIn Vitro Culture Post-fertilization embryo culture took place in modified synthetic oviduct fluid medium (SOF) under paraffin oil in a humidified atmosphere of 5% CO2, 5% O~, 90% N2 at 39°C (4). Twenty-four hours after insemination, presumptive zygotes were denuded by vortexing for 2 min in 2 mL PBS. The zygotes were subsequently washed 4 times in PBS and in SOF before being transferred in groups of 20 to 30 to the culture droplets (1 zygote per microliter medium). Fetal calf serum was added to the droplets (10% v/v) 24 h aider placement in culture (i.e., 48 h post insemination). Cleavage was assessed 48 h after placement in culture, i.e., 72 h post insemination (percentage of noncleaved, 2 to 4 cells, 5 to 8 cells). The number of embryos developing to at least the expanded blastocyst stage was assessed on Days 6 and 8 of culture (i.e., Days 7 and 9 post insemination). Hatching was recorded on Day 8 of culture and expressed as a percentage of Day 8 blastocysts. For the estimation of total cell numbers at Day 8 of culture, blastocysts were placed on a slide, air-dried and fixed in ethanol (100%) overnight. They were subsequently stained using Hoechst 33342 (10 mg/mL in 2.3% sodium citrate, w/v) and visualized with an epifluorescence microscope (Zeiss, Oberkochen, Germany). Statistical Analysis Treatment effects on cleavage rate, percentage of 5 to 8 cell embryos, blastocyst rate and blastocyst hatching rate were determined by examining totals of all replicates using Chisquare analysis or Fisher's exact test. Cell numbers were expressed as mean+SD, and were compared with the nonparametric Mann-Whitney test. A P value less than 0.05 was considered to be statistically significant. ~S~TS Experiment 1 The results of Experiment 1 are shown in Table 1. There were no differences in cleavage rates between treatments. However, the percentage of 5 to 8 cell embryos at 72 h post insemination was significantly lower following CX treatment than the control (P<0.02), irrespective of the duration of exposure to the inhibitor (64 vs 42 to 51% for control and CX treatments, respectively). This was reflected in a lower rate of blastocysts at Day 6 for all CX-treated oocytes (31 vs 9 to 15% for control and CX treatments, respectively; P<0.002). While the blastocyst rate at Day 8 was always lower in CX-treated oocytes, the value was only significantly lower than in the control when CX was present for longer than 12 h. A marked decrease in development was noted following inhibition for 18 h or more compared with that ofunblocked oocytes (40 vs 17 to 19% for control and CX treatments, respectively; P<0.0002). There were no significant differences in hatching rates among any of the treatments. Only oocytes treated with CX for 18 h had a significantly lower blastocyst cell number than the control (84+6 vs 113+8, P<0.04).

176

170

151

173

CX 6hb

CX 12h

CX 18h

CX 24h

146

123

150

155

120

n

84

81

88

88

90

%

Cleavage rate

73

64

86

83

86

n

42 e

42 e

51 e

47 e

64 d

%

5-8 cells

15

23

19

15

42

n

9e

15 e

11 e

9e

31 d

%

Day 6 c

17f

19ef

29 d

29 d

40 d

%

7

6

13

22

20

n

24

21

27

43

38

%

Hatching

95_+6 (28) de

84+6 (26) e

110+7 (39) d

115+8 (37) d

113+8 (40) d

mean+SEM (n)

Cell number

c Day 6 and Day 8 refer to days of culture. d,e f ' Values in the same column with different superscripts differ significantly (P<0.05). Four replicates carried out.

bl0p, g/mL cycloheximide diluted in M199. Treatment with CX was followed by 24 h of maturation under control conditions.

29

29

49

51

53

n

Day 8c

Blastocyst rate

acontrol oocytes were cultured for 24 hours in M199+10% FCS (v/v).

134

n

Control a

Treatment

Table 1. Effect of inhibition o f protein synthesis for varying intervals from the onset of in vitro maturation on subsequent bovine embryo development

t~

t~ tt3

¢x ro ro

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423

Experiment 2 The results of Experiment 2(a) are presented in Figure l(a). Addition of 40 or 400 ng/mL FSH to M199 stimulated cumulus expansion and significantly increased the cleavage rate (71 vs 83 and 93% for M199, FSH 40 and 400 ng, respectively; P<0.05) and the percentage of oocytes reaching the 5 to 8 cell stage by 72 h post insemination (42 vs 54 and 65%; P<0.05) relative to the control. In addition, the blastoeyst rate at Day 8 of culture was significantly higher for oocytes matured in the presence of FSH alone, irrespective of concentration (27 vs 41 and 44% for M199, FSH 40 and 400 ng, respectively; P<0.05). There were no differences in hatching rates. The addition of FSH to ooeytes in the presence of CX had no effect on any of the parameters studied after removal of the inhibitor and subsequent maturation under control conditions, irrespective of FSH concentration used. The results of Experiment 2(b) are presented in Figure l(b). The presence of FCS during in vitro maturation resulted in a significantly higher cleavage rate (73 vs 90°/,; P<0.0002), % 5 to 8 cells (47 vs 64%; P<0.003) and significantly more blastoeysts at Day 6 (18 vs 31%; P<0.005) and Day 8 (27 vs 40%; P<0.02) of culture than maturation in M199 alone. This is a consistent observation in our laboratory (21). While both the hatching rate and blastocyst cell number were higher in the presence of FCS, the difference was not significant. Following meiotic inhibition in the presence of CX and subsequent culture, the cleavage rate was higher than in M199 alone (73 vs 84 and 88% for M199, CX and CX+FCS, respectively), but compared with MI99+FCS, there was no difference. The blastoeyst rate was significantly lower following CX treatment (see Figure 1). The presence of serum during the 24-h period of CX-induced arrest did not affect any of the parameters studied compared with inhibition in serum-free medium. Experiment 3 The results of the third experiment are shown in Table 2. All oocytes treated with CX were cultured under conditions conducive to maturation for 24 h (i.e., M199+FCS). Thus two controls were used: oocytes cultured in M199+FCS for 24 h (Group 1) and 30 h (Group 6). While there was no difference in cleavage rates between these 2 controls (81 vs 83%), the percentage of 5 to 8 cell embryos at 72 h post inseminationwas significantly lower following 30 h of maturation (44 vs 60%; P<0.002). This was reflected in a lower blastocyst rate at Day 6 (17 vs 29%; P<0.006) and Day 8 (26 vs 40%; P<0.005). Treatment with CX for 6 h at the initiation of culture (Group 2) did not affect the cleavage rate or the percentage of embryos at the 5 to 8 cell stage at 72 h post insemination compared with the 24 h control. The blastocyst rate at Day 6 was significantly lower than the 24 h control (16 vs 29%; P<0.004). The difference at Day 8 was no longer significant (31 vs 40%). These results are consistent with the findings of Experiment 1. Compared with the 30 h control, the 6-h treatment with CX reduced the percentage of 5 to 8 cell embryos at 72 h post insemination (56 vs 44%; P<0.02), but there was no significant difference in the blastocyst rate at Day 6 (16 vs 17%) or Day 8 (31 vs 26%). Treatment with CX after the oocytes had been allowed to mature for varying periods (6, 12 and 18 h for Groups 3, 4 and 5) resulted in a decrease in cleavage and blastocyst rates at

183

192

185

192

179

6 hours CX b, 24 hours S

6 hours S, 6 hours CX, 18 hours S

12 hours S, 6 hours CX, 12 hours S

18 hours S, 6 hours CX, 6 hours S

30 hours S

148

126

134

156

153

151

n

78

66

70

97

102

111

n

44 e

34 e

38 e

51 d

56 d

60 d

%

5-8 cells

30

22

20

32

30

54

n

17e

11 e

11 e

17e

16e

29 d

%

Day 6 c

46

39

27

52

56

74

n

26 e

20 ef

15 f

27 e

31 d

40 d

%

Day 8c

Blastocyst rate

d'e'fvalues in the same column with different superscripts differ significantly (P<0.05). Five replicates carried out.

c Day 6 and Day 8 refer to days of culture.

83 d

66 e

72 e

81 d

84 d

81 d

%

Cleavage rate

bCX: 10~tg/mL cycloheximide diluted in M199.

as: M199+10% FCS (v/v).

186

n

24 hours S a

Treatment

% 54 45 37 48 59 54

n 40 25 19 13 23 25

Hatching

134_+10(40)

131_+10(33)

114_+11(22)

116+8 (48)

128+8 (51)

138+7 (68)

mean+SEM (n)

Cell number

Table 2. Effect of inhibition of protein synthesis for a 6-hour period during in vitro maturation on subsequent bovine embryo development

q~

¢b

2

Thenogeno~gy

425

50

4O

4@

30

2,0 m d~

m

10

10

o

o

(a)

~

÷

~+

~

(b)

Figure I. Effect of ooeyte exposure to (a) FSH (n=580) or (b) FCS (n=659) during cycloheximide (CX)-induced meiotic maturation on subsequent blastocyst development. Blastoeyst yields are relative to oocytes inseminated. Bars with different letters are significantly different (P<0.05). Following treatment with cyeloheximide, ooeytes were cultured for 24 hours in M199+FCS. Days 6 and 8 of culture compared to the 24-h control (Table 2). The most significant decrease in development occurred when oocytes were cultured for 12 h before exposure to CX (15 vs 40%; P<0.0001). Similarly, compared with the 30-h control, the only significant difference in blastoeyst yield occurred when oocytes were cultured for 12 h before exposure to CX (15 vs 26%; P<0.009). There were no differences in hatching rates or blastocyst cell numbers between any of the groups. DISCUSSION The importance of the timing of protein synthesis relative to meiotic resumption for subsequent ooeyte development has been illustrated using inhibitors of transcription (aamanitin, 13, 14), translation (CX, 14, 23) and post-translational modifications (6dimcthylaminopurine, 10, 23, 34). These products disrupt nuclear progression and normal oocyte development, providing evidence for the synthesis and activities of proteins regulating ooeyte maturation.

426

Theriogenology

Cycloheximide, which inhibits peptidyl transferase, suppresses oocyte maturation by blocking synthesis of stage-specific proteins. A variable period of protein synthesis is required in order for oocyte meiotic resumption to occur in several species. This period appears to be related to the time needed for GVBD during IVM. Mouse oocytes take 12 h to mature, and GVBD occurs within 2 h (6). Treatment with CX from the onset of culture does not prevent GVBD (9, 28). Bovine oocytes take 24 h to mature, and GVBD occurs within 6 to 9 h (23). Exposure to CX until 6 h after the onset of IVM prevents GVBD most bovine oocytes (14, 23, 39, 43). Porcine oocytes take 44 h to mature, and GVBD occurs al~er approximately 16 to 20 h (29). When CX was added after 6, 12 and 16h of culture, GVBD occurred in 15, 46 and 75% of the oocytes (9). Similar findings have been reported in horses (3) and goats (19) where incubation in the presence of CX (20 mg/mL) prevented GVBD in all oocytes when present at 2 h from the start of culture. By delaying addition of the inhibitor for progressively longer periods after the resumption of meiosis, it was established that new protein synthesis during the first 8 h of culture is essential for GVBD in bovine oocytes (14, 43). In the study ofKastrop et al. (14), addition of CX at 4, 6 or 8 h after the start of culture resulted in 12, 38 and 81% GVBD, respectively. However, none of the ooeytes reached MII. Only those oocytes cultured for at least 12 h in the absence of the inhibitor reached MII; after 16 h, addition of CX did not affect the proportion reaching MII. In our laboratory, in CX-ffee medium, GVBD occurs in most oocytes (approximately 80% in M199 alone and >95% in M199+FCS), with approximately 65% progressing to MII in M199 alone and >90% in M199+FCS (23). The presence of CX for 24 h blocks these processes, even at low concentrations of the inhibitor (lgtg/mL). The inhibitory effect of CX is fully reversible, both in terms of oocytes undergoing GVBD and their ability to progress to MII (23, 37). However, when the definition of reversibility is broadened to include ability to progress to the blastocyst stage, CX-treated oocytes while exhibiting normal rates of cleavage were notably inferior to the nontreated controls in terms of development to the blastocyst stage (present study and 23). We have extended our previous results by examining the sensitivity of oocytes to exposure to CX over increasing periods before IVM on development in order to explain and improve upon the low development results observed after inhibition (Experiment 1). We observed that exposure to CX for more than 12 h before IVM is sufficient to effect a significant decline in developmental ability compared with that of nonblocked oocytes, the effect being more marked atter longer incubation periods. Although the blastocyst rates decrease, the embryos that do attain the blastocyst stage have a comparable number of cells as their nonblocked counterparts. This is consistent with the results of Saeki et al. (36), who reported live births after the transfer of embryos originating from CX-blocked oocytes. The decline in developmental rates observed in CX-blocked oocytes may be partly due to the fact that in oocytes treated for 24 h with CX, removal of oocytes from CX-supplemented medium results in acceleration of GVBD (cattle: 37; sheep: 26, 30, goat: 19, pig: 18), and a certain degree of chromatin condensation occurs, indicating that some maturation processes are not blocked.

Theriogenology

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Figure 2. Light micrograph of bovine cumulus oocyte complexes (COCs) following exposure to cycloheximide for 24 hours (a) and following a second 24 hours of culture in the absence of the inhibitor (b). Note the intact germinal vesicle in (a) and the expansion of the cumulus and first polar body extrusion in (b). The COCs were fixed in Karnowsky's fixative and individually embedded in 4% Agar. They were post fixed in OSO4 in 0.1M cacodylate buffer, dehydrated, uranyl stained, embedded in Epon and serially sectioned (2 nm). In mice, it has been reported that the choice of culture medium can have a profound impact on spontaneous and ligand-induced oocyte maturation, as well as the efficacy of meiotic inhibitors (7). In Experiment 2, we exposed oocytes to FSH during the period of inhibition in order to improve the cytoplasmic maturation of the oocytes without altering their nuclear status. Van Tol et al. (45) reported that meiotic resumption in vitro of bovine oocytes from 2 to 8 mm follicles maintained in meiotic arrest by the membrana granulosa is triggered by FSH and not by LH, this being supported by the fact that receptors for FSH but not for LH are transcribed in the cumulus and granulosa cells of such follicles. In our present study, FSH did not overcome the inhibitory effect of CX, and its addition to CX-inhibited oocytes had no affect on subsequent development. This is similar to the findings of Saeki et al. (36). Similarly in Experiment 2, we did not find any difference between inhibition in the presence or absence of serum. These findings are perhaps not unexpected in light of our previous results using epidermal growth factor (22, 23). However, FSH, FCS and EGF are capable of stimulating development in unblocked oocytes (present results, 21, 23). It is clear therefore that the method of meiotic arrest plays a major role in the ability of blocked oocytes to respond to putative stimulatory substances. The 2 metaphases during bovine oocyte nuclear maturation (MI at about 12 h and MII at about 20 to 24h after initiation of culture) are correlated with biphasic appearances of high MPF activity in the cytoplasm (46). Both increases in MPF activity during oocyte maturation require active protein synthesis and phosphorylations (20, 24). Wu et al. (47) noted that protein synthesis in the bovine oocyte is maximal during the initial 12 h of IVM. In our

428

Thenogenology

system, most of the oocytes (>90%) cultured in the presence of serum were at MI at 12 h after initiation of culture (23). Moreover, those oocytes in which protein synthesis was blocked by exposure to CX after 12 h of culture were the most susceptible in terms of subsequent development. It is also important to highlight that 30 h of maturation resulted in a significant reduction in embryo development compared with the standard 24 h culture period, undoubtedly due to the aging of oocytes. While exposure to CX did, in general, lead to a reduction in development compared with that of oocytes matured for 30 tl, in most cases the difference was not significant. Thus, decreased development would seem at least in part to be due to prolonged culture and not to the detrimental effect of CX exposure. This explanation would be consistent with the finding in Experiment 1 that 6-h of exposure of oocytes to CX was not detrimental compared with the control treatment. Using physiological and pharmacological methods, various authors (1, 2, 5, 10, 12, 17, 32, 33, 36, 38, 40) have reported on the maintenance of meiotic arrest in bovine oocytcs. However, almost none of their studies report on developmental rates of oocytes following periods of blockage. Furthermore, many of the products used in the above studies are not compatible with the long-term survival of oocytes and therefore cannot be used to enhance developmental competence. While encouraging and indicative of the feasability of in vitro meiotic inhibition as a tool in the study of the mechanisms involved in competence acquisition, the results presented in our study are still inferior to those obtained with unblocked oocytes in terms of oocytes attaining the blastocyst stage. A system capable of reproducing the developmental rates observed in nonblocked control oocytes would open up the possibility of using this system on groups of less competent oocytes e.g., growing oocytes or those from prepubertai animals (15) to increase their developmental competence. A system based more closely on the physiological situation, perhaps using follicle cells, would better approximate the situation in vivo. However, a reliable system capable of yielding blastocyst rates similar to those of unblocked oocytes has yet to be established. Another observation from this study is the importance of choosing appropriate endpoints for experiments. It is clear from the present results that MII and cleavage rates were only indicative of but not sufficient to differentiate between potentially low and high yielding groups of oocytes/treatments. It has been well established that culture conditions for in vitro maturation of mammaiian oocytes can significantly influence the subsequent development of such oocytes (mouse: 44; pig: 48; cattle: 15, 35). In conclusion, the present study demonstrates that bovine oocytes in which meiotic arrest is maintained by CX retain developmental competence following reversal of arrest and submission to in vitro maturation, fertilization and culture. Inhibition with CX results in normal cleavage rates following reinstatement of conditions conducive to maturation, with a reduction in final blastocyst yields compared with that of unblocked oocytes. It would seem that inhibition of meiotic resumption in vitro with CX for periods longer than 12 h irreversibly disrupts some processes necessary for full developmental competence. The high sensitivity of such oocytes to CX would indicate that specific proteins necessary for GVBD and subsequent development are not present in the cytoplasm of fully grown immature bovine oocytes. It is probable that during the final few days before ovulation, during which time the follicle grows to 15 mm, such proteins are synthesized.

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