THE JOURNAL OF EXPERIMENTAL ZOOLOGY 258~95-103 (1991)
Neomycin, an Inhibitor of Phosphoinositide Hydrolysis, Inhibits the Resumption of Bovine Oocyte Spontaneous Meiotic Maturation SHERYL T. HOMA Department of Zoology, Arizona State University, Tempe, Arizona 85287 The possibility that the intracellular signals generated upon phosphoinositide ABSTRACT hydrolysis are involved in regulating bovine oocyte spontaneous meiotic resumption was investigated. Oocytes were mass-harvested and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin in the presence or absence of neomycin (an inhibitor of phosphoinositide hydrolysis) or phorbol myristate acetate (an activator of protein kinase C). The role of intracellular calcium was examined by preloading with BAPTAIAM (a calcium chelator) prior to culture. Meiotic maturation was scored cytogenetically. 1)Neomycin induces an irreversible inhibition of germinal vesicle breakdown which does not exceed 60% and is apparent at concentrations of 5 mM or above. Progression of meiosis past metaphase I is inhibited at concentrations of 2.5 mM or above. The full effect of neomycin is only apparent if it is presented to the oocytes within 3 h of follicular release, although germinal vesicle breakdown is not observed until 9 h culture under control conditions. 2) PMA alone has negligible effect on germinal vesicle breakdown, but it acts synergistically with 2 mM IBMX to inhibit this process. PMA has a dual effect on the progression of meiosis past metaphase I: 1nM PMA has a stimulatory effect while 1 pM PMA blocks the ability of oocytes to reach anaphase I or beyond. These observations are not found with a non-tumor-promoting phorbol ester. 3) Spontaneous meiotic resumption is not significantly affected in the absence of added exogenous calcium. However, oocytes preloaded with BAPTA/AM exhibit a dose-dependent inhibition of germinal vesicle breakdown, even in the presence of extracellular calcium. These results provide indirect evidence for a role for phosphoinositide metabolism in bovine oocyte maturation; it is suggested that intracellular calcium mobilization is prerequisite for meiotic resumption and protein kinase C may modulate CAMP-dependent inhibition of this response.
Resumption of meiotic maturation in mammalian oocytes is triggered by luteinizing hormone in vivo (Tsafriri, '78). However, the biochemical mechanisms which mediate this process have yet t o be clearly defined. If oocytes are removed from their follicular environment, they will spontaneously mature through all stages of meiosis in culture under defined conditions (Tsafriri, '78). It has been clearly established that elevations in cyclic adenosine monophosphate (CAMP) can inhibit spontaneous meiotic maturation in several mammalian species (Cho et al., '74; Wasserman et al., '76; Magnusson and Hillensjo, '77; Dekel and Beers, '78; Rice and McGaughey, '81; Ball et al., '83; Racowsky, '86; Homa, '881, while increasing evidence supports a central role for calcium in both the resumption and progression of meiosis (Batta and Knudsen, '80; Paleos and Powers, '81; Jagiello et al., '82; Maruska et al., '84; Bae and Channing, '85; Racowsky, '86).It has been demonstrated that calcium levels in intact rat oocytes increase concomitantly with increases in serum0 1991 WILEY-LISS, INC.
luteinizing hormone (Batta and Knudsen, '80) and therefore prior t o initiation of meiotic maturation. In addition, studies on hamster oocytes have revealed that calcium can override meiotic arrest maintained by the cAMP analogue dibutyryl cAMP (Racowsky, '86). It is widely accepted that stimulus-receptoractivated responses which are accompanied by changes in calcium are initiated by membrane phosphoinositide hydrolysis (Michell, '86; Berridge and Irvine, '89; Williamson and Monck, '89). This results in the production of two intermediates: diacylglycerol, which activates protein kinase C (Nishizuka, 'SS), and inositol trisphosphate, which interacts with receptors on the endoplasmic reticulum resulting in mobilization of calcium from within these stores (Berridge and Irvine, '89). While it is known that luteinizing hormone stimulation results in elevated levels of Received March 2, 1990; revision accepted August 2, 1990.
96
S.T. HOMA
CAMP,more recently it has been demonstrated that luteinizing hormone may also activate phosphoinositide turnover in ovarian cells, resulting in an elevation of inositol trisphosphate (Davis et al., '86, '87; Dimino et al., '87; Sadighian et al., '89). Furthermore, protein kinase C, which has been shown to affect CAMP formation in rat (Shinohara et al., '85) and human (Jalkanen et al., '87) granulosa cells, has been characterised in ovarian tissue in both the bovine corpus luteum (Davis and Clark, '83; Noland and Dimino, '86) and in porcine granulosa (Veldhuis and Demers, '86) and luteal (Wheeler and Veldhuis, '89) cells. From the evidence presented above, it appears that phosphoinositide metabolism may play a significant role in mediating the resumption of mammalian oocyte maturation. Neomycin, a polycationic aminoglycoside antibiotic, binds with high affinity to phosphatidylinositol 4,5bisphosphate (Schacht, '78; Gabev et al., '891, thereby interfering with its subsequent hydrolysis and generation of second messengers (Downes and Michell, '81; Tysnes et al., '88; Whitaker, '89). In this study, bovine oocyte spontaneous maturation was examined in the presence of neomycin. In addition, the role of protein kinase C and calcium in meiotic resumption was also investigated. The former was studied by using phorbol myristate acetate (PMA), which mimics the action of diacylglycerol on protein kinase C activity, while BAPTA/AM, a potent calcium chelator which can be trapped inside the cell following deesterification of the acetoxymethyl ester (AM), was used to investigate the latter.
cal Co., St. Louis, MO) were prepared in 2ABMOC medium containing 0.5% bovine serum albumin (fraction V, Sigma Chemical Co.). Stock solutions of phorbol 12-myristate 13-acetate (PMA) (1mM) and 4a-phorbol 12,13-didecanoate (1 mM) (Sigma Chemical Co.) and BAPTA/AM (10 mM) (Calbiochem., La Jolla, CA) were prepared in DMSO such that addition t o the culture medium resulted in a final concentration of 0.2% DMSO. In experiments in which phorbol esters or BAPTA/AM treatment was examined, an equivalent volume of DMSO was added to control cells. For treatment with neomycin, cycloheximide, IBMX, or phorbol esters, oocytes were thoroughly washed in their respective culture medium prior t o incubation. For treatment with BAPTA/AM, groups of 24-36 oocytes were collected in 2ABMOC medium, washed briefly in 150 mM NaCl + 25 mM HEPES, pH 7.2, and then incubated in 2 ml of this medium for 30 min in the presence or absence of BAPTA/AM at 38°C. The loaded oocytes were washed 3 times in 2 ml 2A-BMOC medium containing 0.5%bovine serum albumin t o remove exogenous BAPTA/AM, and then resuspended in 2A-BMOC for long-term culture. Oocytes were cultured in 0.2 ml sterile 2A-BMOC medium, sometimes in the presence of 0.5% bovine serum albumin, and were incubated in wells of sterile Lab-Tek tissue culture chamber slides (#4838, Miles Scientific, Naperville, IL) at 38°C in a humidified atmosphere of 5% C02 + 5% O2 + 90% Nz. Groups of cells (8-12) were treated in duplicate or triplicate for each experiment and each experiment was repeated at least 3 times.
METHODS Collection of oocytes Reproductive tracts from pregnant and nonpregnant cows were obtained from a local slaughterhouse. Ovaries were removed immediately following slaughter. Oocytes devoid of surrounding cumulus cells were purified in chemically defined culture medium, 2A-BMOC, by using a mass harvesting procedure described previously (Homa, '88). Oocytes were then selected for culture with the aid of a light microscope by using established criteria (Home, '88). The average collection time of oocytes from the midpoint of follicular release (0 h) to the start of culture was 1 h.
Determination of meiotic stage Oocytes were fixed, air-dried, and stained (McGaughey and Polge, '71). Oocyte maturation was scored cytogenetically according to criteria previously described (McGaughey et al., '79; Homa, '88). Statistical analyses were carried out by using Student's 't' test.
Culture conditions Sterile solutions of neomycin sulphate (0-10 mM), cycloheximide (10 pg/ml), and 3-isobutyl-lmethyl xanthine (IBMX) (2 mM) (Sigma Chemi-
Protein synthesis Oocytes were cultured in 0.2 ml 2A-BMOC medium containing 0.5% bovine serum albumin, in the presence or absence of either 5 mM neomycin or 10 pg/ml cycloheximide, with 2.5 pCi/ml [U14Clglycine (96 mCi/mmole, New England Nuclear). Three replicates were prepared for each treatment group, containing 20 oocytes per replicate. Incubations were initiated within 3 h of follicular release and terminated after 10 h following follicular release. Incorporation of radio-
NEOMYCIN AND MEIOSIS c
100
-
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9
Neornycin (mM)
Fig. 1. Dose response of meiotic maturation to neomycin. Bovine oocytes were cultured for a total period of 24 h following release from the ovary. Each value represents the mean of 5-12 replicate determinations t S.E.M.
activity into TCA-soluble and -insoluble material was determined as described by Stern et al. ('72).
RESULTS Effect of neomycin on spontaneous meiotic maturation About 90% bovine oocytes devoid of surrounding cumulus cells will spontaneously mature following 24 h in culture as shown in Figure 1. Sixty-nine percent of these maturing oocytes have progressed past metaphase I at this time. In the presence of 5 mM neomycin, there is a 47% inhibition of germinal vesicle breakdown (untreated vs. 5 mM neomycin-treated P < 0.001). This level of inhibition is maintained up to 60% at higher doses of neomycin. At lower concentrations which do not inhibit germinal vesicle breakdown, the proportion of oocytes reaching anaphase I or beyond is significantly reduced (untreated vs. 2.5
97
mM neomycin-treated % anaphase I to metaphase I1 P < O.OOl), while at concentrations of 5 mM or greater, progression of meiosis past metaphase I is almost totally prevented. As neomycin was presented t o the oocytes in the form of a sulphate complex, a dose response t o sodium sulphate up t o 30 mM concentration was carried out. The experiments revealed that sodium sulphate had no significant effect on meitoic maturation (data not shown), suggesting that the effects observed in the presence of neomycin sulphate are attributed t o neomycin. It could be argued that the effects of neomycin on bovine oocyie maturation may be due to the ability of the antibiotic t o inhibit protein synthesis. In the absence of any treatment, germinal vesicle breakdown has occurred in the majority of cultured bovine oocytes by 10 h following release from the ovary (Homa, '88; Sirard et al., '89). 00cytes cultured with as low a concentration as 10 pg/ml cycloheximide for the same period of time demonstrate a significant inhibition of protein synthesis as shown in Table 1(control vs. treated; P < 0.001). Furthermore, 10 pg/ml cycloheximide blocks germinal vesicle breakdown as well as subsequent steps during meiotic maturation of bovine oocytes (Sirard et al., '89). While incubation with 5 mM neomycin causes a significant inhibition of germinal vesicle breakdown (Fig. l),in contrast t o cycloheximide, it failed to inhibit protein synthesis under identical culture conditions (Table 1).Thus the inhibitory effects of neomycin on germinal vesicle breakdown are unlikely to be due to protein synthesis inhibition. The effect of neomycin on germinal vesicle breakdown is not reversible at any dose examined (Fig. 21, as washing oocytes pretreated for 24 h with neomycin and then culturing for an addi-
TABLE 1. Effect of cycloheximide and neomycin on incorporation of [U-I4 Clglycine into oocyte proteins'
Contro1 TCA insoluble (cpm/lO oocytes) TCA soluble ( c p d l 0 oocytes) % TCA insoluble of total uptake % [U-'*C]glycine uptake from medium
*
Cycloheximide (10 pgiml)
* 18"
316
?
96
237
1866
?
181
461 t 35*
170
2184 t 135
2387
17.43 t 0.46**
6.66 t 0.41**
0.658
k
0.032
Neomycin (5 mM)
?
0.621 t 0.051
14.09 t 2.85 0.554 t 0.064
'Values are the mean S.E.M. of three replicate samples. Numbers with the same superscript are significantly different. *, ** P < 0.001.
S.T. HOMA
98 100
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w
201
0-
10
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0
0%
0
1
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Neomycin (mu)
Fig. 2. Reversibility of neomycin effect. Oocytes were cultured for 24 h in increasing concentrations of neomycin, subsequently washed 3 times, and then incubated for a n additional 24 h in the absence of neomycin. Values are the mean of 6-12 replicate determinations t S.E.M.
w 0
2
4
6
8
10 12 14 16 18 20 22 24
Time in control medium (h)
Fig. 3. Determination of the period of sensitivity to neomycin. Bovine oocytes were removed from ovaries at 0 h and cultured in control medium. At intervals of time, groups of 30 oocytes were resuspended in 5 mM neomycin. Incubations were continued for a total of 24 h. Each value represents the mean of 9-13 replicate determinations 2 S.E.M.
tional24 h in the absence of neomycin results in a and an elevation in intracellular levels of calsimilar dose response pattern as treatment with cium, it appeared necessary t o investigate which neomycin for 24 h only. To determine whether of these pathways could influence meiosis. Geroocytes became insensitive t o neomycin treat- minal vesicle breakdown is unaffected by increasment after a period of time in culture, they were ing doses of PMA, which activates protein kinase incubated in control medium for increasing pe- C; however, it has a dual effect on the progression riods of time and then transferred to medium con- of meiosis past metaphase I (Fig. 4a); 1nM PMA taining 5 mM neomycin for a total incubation augments the proportion of oocytes reaching time of 24 h. Figure 3 shows that for over 2 h anaphase I to metaphase I1 (untreated vs. 1 nM following follicular release, oocytes are sensitive PMA-treated P < 0.05) while higher concentrato neomycin treatment, as only 32.4% have tions of the phorbol ester significantly inhibit the undergone germinal vesicle breakdown by the ability of oocytes t o progress beyond metaphase I end of the culture period. However, the ability of 5 (untreated vs. 1 pM PMA-treated P < 0.001). Interestingly, the phorbol ester exerts a dosemM neomycin to inhibit germinal vesicle breakdown is significantly reduced (P < 0.001) by al- dependent synergistic effect on inhibition of germost 50% if it is added at 4 h after release from minal vesicle breakdown in the presence of the follicle. There is a 2 h lag period between 4 IBMX, an inhibitor of CAMP phosphodiesterase and 6 h after follicular release, until the remain- (Fig. 4b). In this study it can be seen that oocytes ing oocytes become insensitive to neomycin. Inhi- will mature in the presence of PMA or 2 mM bition of germinal vesicle breakdown by 5 mM IBMX alone, even though the proportion of matneomycin is abolished if neomycin is presented to uration is significantly decreased in the presence the oocytes at 7 h or more following follicular re- of 2 mM IBMX compared with untreated controls lease. It should be noted that it is not until 9 h (P < 0.02) as previously reported (Homa, '88). following follicular release that bovine oocytes ac- However, in the combined presence of 2 mM tually demonstrate germinal vesicle breakdown IBMX and PMA, the rate of germinal vesicle under control culture conditions in 2A-BMOC breakdown significantly declines at the highest medium in the absence of neomycin (Homa, '88). dose of PMA tested (2 mM IBMX vs. 2 mM IBMX + 1 pM PMA, P < 0.001). IBMX almost comPhorbol ester and spontaneous maturation pletely inhibits the progression of meiosis past One interpretation for the observation that metaphase I and this effect is unaltered in the neomycin inhibits meiotic resumption could im- presence of PMA. The specificity of the effects of PMA was invesplicate a role for phosphoinositide turnover in this process. As hydrolysis of phosphoinositides re- tigated by using similar concentrations of the sults ultimately in activation of protein kinase C non-tumor-promoting phorbol ester, 4a-phorbol
99
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Phorbol ester
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-
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O
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-- -A. 6
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Fig. 4. Dose response of meiotic maturation to PMA. 00cytes were incubated with increasing concentrations of PMA (circles) or 4a-phorbol 12J3-didecanoate (triangles) a) in the absence of b) in the presence of 2 mM IBMX for 24 h. Values represent the mean of4-9 replicate determinations 2 S.E.M.
12,13-didecanoate.Figure 4 shows that this phorbol ester has no significant effect either on germinal vesicle breakdown or on the progression of meiosis. Furthermore, unlike PMA, this phorbol ester is inactive in the presence of IBMX.
Calcium and spontaneous maturation The effects of changes in intracellular calcium levels on bovine oocyte meiotic maturation were monitored indirectly by using the calcium chelator BAPTA/AM. The calcium probe was trapped inside oocytes within 90 min of release from the follicle. The oocytes were subsequently washed and incubation was continued in fresh control medium. Figure 5 shows that oocytes preloaded with BAPTA/AM remain at the germinal vesicle stage following 12 h incubation whereas
Fig. 5. Dose response of germinal vesicle breakdown to BAPTMAM treatment. Oocytes were loaded with BAF’TAi AM 1 h following follicular release, then cultured until 12 h following follicular release. Each value represents the mean of 3-8 replicate determinations 2 S.E.M.
untreated cells have reached diakinesis at this time. This inhibition of meiotic resumption is dose dependent, and is evident at concentrations of 10 p M BAPTAiAM or higher (0 vs. 10 p M ,P < 0.02; 0 vs. 20 pM, P < 0.001). These effects are unlikely due to the accumulation of potentially toxic acetoxymethyl esters as the inhibition induced by BAPTA/AM is transient. Table 2 shows that following 24 h culture, the effect of BAPTA/ AM is no longer apparent. It is possible that the BAPTA within the cell becomes saturated with calcium with the constant exchange from the extracellular environment after a period of time, thereby allowing levels of intracellular calcium t o become elevated. By re-treating the oocytes with BAPTA/AM for a second time after 12 h incubation (Table 21, inhibition of meiotic resumption is maintained following 24 h culture (treated vs. untreated P < O.OOl), indicating that prevention of germinal vesicle breakdown by inhibition of elevation of intracellular calcium is not a transient effect. To determine whether the effect of BAPTA is due to inhibition of mobilization of intracellular calcium from intracellular stores, or whether it is due to prevention of calcium movement through the plasma membrane, oocytes preloaded with BAPTA/AM were cultured in the presence of negligible exogenous calcium. Table 3 shows that germinal vesicle breakdown will occur in the absence of added exogenous calcium. Although the proportion of oocytes resuming meiosis under these conditions is reduced from 67.8% in medium routinely supplemented with calcium (1.7 mM) t o
100
S.T. HOMA T A B L E 2. Long-term effects of BAPTAIAM treatment' Experiment A B
Treatment Control 20 (LMBAPTMAM Control 20 LLMBAPTMAM
% Germinal vesicle breakdown
80.4 ? 95.8 ? 96.7 T+_ 15.9 &
16.0 (3) 5.1 (3) 3.6 (6)* 5.5 16)"
'In experiment A, oocytes were treated with or without BAPTAiAM 1 h following follicular release, while in experiment B, oocytes were treated at 1 h and a t 12 h following follicular release. Oocytes were cultured for a total time of 24 h. Values represent the mean i- SEM. Numbers in parentheses represent numbers of replicates. *Significantly different, at P < 0.001.
T A B L E 3. Effect of exogenous calcium on meiotic maturation of oocytes pretreated with or without 20 p,M BAPTAIAM'
peting with calcium for channel binding (Hughes et al., '88; Dulon et al., '89). In addition, it can affect calcium mobilization by chelating inositol % Germinal Vesicle Breakdown trisphosphate in permeabilised cells (Prentki et of Total Oocytes al., '86). However, the effect of neomycin on oocyte CaCb [FMI Untreated Treated maturation is unlikely attributed to its properties 0 45.8 ? 7.2 (7) 7.9 ? 6.5 (7) as a calcium antagonist. Firstly, removal of ex1 45.3 k 4.3 (7) 5.9 ? 4.5 (7) tracellular calcium had no effect on bovine oocyte 10 42.6 ? 8.3 (7) 5.2 ? 4.0 (7) maturation. Secondly, aminoglycoside antibiotics 100 64.9 ? 4.4 (7) 13.2 k 6.7 (7) do not readily penetrate cell membranes (Dulon 1,700 67.8 k 10.0 (6) 4.5 ? 3.1 (6) et al., '89). Even if neomycin did enter the cell in 10ocytes were cultured for 12 h following follicular release. Numbers significant quantities, it is unable to inhibit gerin parentheses represent numbers of experiments. Values represent minal vesicle breakdown beyond 60% whereas themean 2 S.E.M. treatment with the calcium chelator BAPTA/AM almost totally abolishes germinal vesicle break45.8% in the identical medium without calcium down. supplementation, this difference is not signifiIt is of interest that total inhibition of germinal cant. Moreover, this reduction does not occur in vesicle breakdown is not obtained with neomycin, the presence of minimal calcium (100 pM), 17 whereas in the presence of BAPTA/AM, meiotic times less than that normally used for culture (1.7 resumption is almost completely abolished. It is mM). Germinal vesicle breakdown is almost to- generally accepted that inositol trisphosphate, tally abolished in calcium-free media if the cells generated upon phosphatidylinositol 4,5-bisphosare preloaded with BAPTA/AM (45.8 t 7.2% phate hydrolysis, is responsible for binding t o rewithout BAPTA/AM t o 7.9 k 6.5%with BAPTA/ ceptors on the endoplasmic reticulum, stimulrtAM). ing release of calcium into the cytosol (Berridge and Irvine, '89). However, there is strong eviDISCUSSION dence t o suggest that calcium is additionally seThe results of these studies clearly show that questered in intracellular pools which are inacthe phospholipase C inhibitor, neomycin, inhibits cessible t o inositol trisphosphate (Berridge and bovine oocyte maturation, and that this effect is Irvine, '89). While neomycin could effectively premost likely mediated by decreased intracellular vent mobilisation of calcium from the inositol calcium mobilisation. Furthermore, activation of trisphosphate-sensitive pool by inhibition of phosprotein kinase C may be important in regulating pholipase C (Downes and Michell, '81; Tysnes et CAMP-maintained meiotic arrest. al., '88; Whitaker, '89), it has not been shown to While it has been accepted that neomycin in- affect inositol trisphosphate-insensitive pools. hibits phosphoinositide hydrolysis (Downes and Thus, if germinal vesicle breakdown requires calMichell, '81; Tysnes et al., '88; Whitaker, '89>,this cium mobilisation from both the inositol trisphospolycationic antibiotic can also effectively block phate-sensitive and -insensitive pools, then calcium influx across the plasma membrane via neomycin may not be expected t o completely abolmechanisms unrelated to inhibition of phos- ish germinal vesicle breakdown. phoinositide hydrolysis, probably by directly comThe effect of neomycin is apparent only if treat-
NEOMYCIN AND MEIOSIS
ment is within 3 h following release of the oocyte from the follicle. Subsequent treatment with neomycin is only effective in about 50% of responding oocytes after 4 h and this degree of sensitivity is maintained for a n additional 2 h. By 7 h following ovarian release, the oocytes have become committed to mature, as treatment with neomycin at this time is completely ineffective, even though bovine oocytes do not undergo germinal vesicle breakdown until 9 h following follicular release (Homa, '88). Similar experiments carried out in the presence of IBMX have demonstrated that about 50% of responding bovine oocytes become committed to mature following 7 h follicular release (Homa, '88), this sensitivity again being maintained for an additional 2 h period, until 9 h from follicular release when the effects of IBMX are abolished. Thus, the pattern of commitment to maturation in the presence of IBMX compared with that in the presence of neomycin is almost identical, with the exception of a 3 h time difference. These results suggest that phosphoinositide metabolism is enhanced prior t o the period at which they become insensitive to increases in levels of CAMP. Hence, CAMPdependent inhibition of meiotic resumption may be overcome by an event mediated by a second messenger(s) derived from phosphoinositide metabolism. As phosphoinositide turnover yields several important second messengers, it was of interest t o determine which of these could be involved in meiotic maturation. It is possible that diacylglycerol is involved in triggering germinal vesicle breakdown as phorbol esters have been shown t o stimulate rat oocyte maturation (Aberdam and Dekel, '85). On the other hand, activation of protein kinase C has been shown t o inhibit this process in mouse oocytes (Urner and SchorderetSlatkine, '84; Bornslaeger et al., '86). Although activation of protein kinase C by phorbol ester does not inhibit bovine oocyte germinal vesicle breakdown, even at relatively high concentrations, it does have a significant effect on the progression of meiosis. This is consistent with observations made by Bornslaeger et al. ('86) who found that phorbol esters impair the formation of the meiotic spindle, such that polar body emission is prevented. The significance of the dual effect of this phorbol ester on meiotic progression in the bovine oocyte is currently unknown, although it may relate t o the positive and negative feedback roles associated with protein kinase C subspecies (Nishizuka, '88).
101
It is of interest that PMA, while having no demonstrable effect on meiotic resumption, acts synergistically with IBMX, an inhibitor of cAMP phosphodiesterase, to inhibit bovine oocyte germinal vesicle breakdown. Recently it has been shown that phorbol ester can enhance the response of adenylate cyclase t o luteinizinghormone stimulation in bovine luteal cells (Budnik and Mukhopadhyay, '871, and it is well established that cAMP plays a significant role in maintaining meiotic arrest. Protein kinase C has been extracted from ovarian tissue (Davis and Clark, '83; Noland and Dimino, '86; Wheeler and Veldhuis, '89>,and activation of this enzyme enhances levels of cAMP in human granulosa cells (Jalkanen et al., '87). It has been suggested that activation of protein kinase C may result in phosphorylation of the inhibitory subunit of adenylate cyclase, thereby rendering that part of the enzyme complex inactive (Jakobs et al., '86). In the presence of PMA and IBMX, this would result in an enhanced increase of cAMP compared with levels in the presence of IBMX or PMA alone, thus promoting inhibition of germinal vesicle breakdown. As it is possible that bovine oocytes have a very active cAMP phosphodiesterase (Homa, '88), treatment with PMA alone may be expected t o have little effect on meiotic resumption. Indeed, when bovine oocytes are treated with either forskolin or 8-bromo-CAMPto raise levels of CAMP,the inhibitory effects on maturation are masked following prolonged culture, probably due to activity of such a phosphodiesterase (Homa, '88). Investigation of the effects of calcium on bovine oocyte maturation yielded different results. Studies with the calcium probe BAPTA/AM strongly suggest that an increase in intracellular calcium is required for meiotic resumption. Similar effects using BAPTA/AM have also been observed in the porcine oocyte (M. Kaufman and S. T. Homa, unpublished data). In addition, as a considerable proportion of oocytes are capable of undergoing germinal vesicle breakdown in the presence of negligible levels or no added exogenous calcium, this supports the hypothesis that it is mobilization of calcium from intracellular stores that is prerequisite for this process. Previous studies have revealed an absolute dependency on calcium for germinal vesicle breakdown under strictly defined conditions in hamster oocytes (Racowsky,'86). Furthermore, there is a relatively large increase in calcium levels concomitant with meiotic resumption in rat oocytes (Batta and Knudsen, '80).
102
S.T.HOMA
Although many of the effects induced by luteinizing hormone are maintained by CAMP, it has recently been shown that this hormone can stimulate phosphoinositide metabolism (Davis et al., '86, '87; Dimino et al., ,871, resulting in an increase in inositol phosphates. This effect is completely abolished upon activation of protein kinase C (Davis et al., '89). However, these responses have been associated with granulosa cells and the corpus luteum, and the exact mechanism of luteinizing hormone at the level of the oocyte is largely unknown. It would be of great interest to determine whether the mechanisms which appear to be associated with phosphoinositide metabolism during spontaneous meiotic maturation can be extended to hormone-induced meiotic resumption in vivo.
(1986) Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Biochem. J., 238:597-604. Davis, J.S., L.L. Weakland, R.V. Farese, and L.A. West (1987) Luteininzing hormone increases inositol trisphosphate and cytosolic free Ca2+ in isolated bovine luteal cells. J. Biol. Chem., 262:8515-8521. Davis, J.S., L.L. Weakland, R.G. Coffey, and L.A. West (1989) Acute effects of phorbol esters on receptor-mediated IPS, CAMP,and progesterone levels in rat granulosa cells. Am. J. Physiol., 256:E368-E374. Dekel, N., and W.H. Beers (1978) Rat oocyte maturation in vitro: Relief of cyclic AMP inhibition by gonadotropins. Proc. Natl. Acad. Sci., USA, 75:4369-4373. Dimino, M.J., J. Snitzer, and K.M. Brown (1987) Inositol phosphates accumulation in ovarian granulosa after stimulation by luteinizing hormone. Biol. Reprod., 39:1129-1134. Downes, C.P., and R.H. Michell (1981) The polyphosphoinositide phosphodiesterase of erythrocyte membranes. Biochem. J., f98:133-140. Dulon, D., G. Zajic, J.-M. Aran, and J. Schacht (1989) Aminoglycoside antibiotics impair calcium entry but not ACKNOWLEDGMENTS viability and motility in isolated cochlear outer hair cells. J . Neurosci. Res., 24:338-346. The author would like to thank Timothy Wyant E., J. Kasianowicz, T. Abbott, and S. McLaughlin Gabev, and Robert Russell for excellent technical assis(1989) Binding of neomycin to phosphatidylinositol 43tance and Dr. Douglas Chandler for reading the bisphosphate (PIP,). Biochim. Biophys. Acta, 979:105-112. manuscript. This research was supported by Homa, S.T. (1988) Effects of cyclic AMP on the spontaneous grant No. HD 20884 from the National Institutes meiotic maturation of cumulus-free bovine oocytes cultured in chemically defined medium. J. Exp. Zool., 248:222-231. of Health . Hughes B.P., A.M. Auld, and G.J. Barritt (1988) Evidence that neomycin inhibits plasma membrane Ca2+ inflow in LITERATURE CITED isolated hepatocytes. Biochem. Pharmacol., 37:1357-1361. Aberdam, E., and N. Dekel (1985) Activators of protein ki- Jagiello, G., M.B. Ducayen, R. Downey, and A. Jonassen (1982) Alterations of mammalian oocyte meiosis I with dinase C stimulate meiotic maturation of rat oocytes. valent cations and calmodulin. Cell Calcium, 3:153-162. Biochem. Biophys. Res. Commun., 132.570474. Bae, I.H., and C. Channing (1985) Effect of calcium ion on the Jakobs, K.H., Y. Watanabe, and S. Bauer (1986) Interactions between the hormone-sensitive adenylate cyclase system maturation of cumulus-enclosed pig follicular oocytes and the phosphoinositide-metabolizing pathway in human isolated from medium-sized Graafian follicles. Biol. Replatelets. J. Cardiovasc. Pharmacol., 8(Suppl. 8):S61-S64. prod., 33:79-87. Ball, G.D., R.W. Lenz, R.L. Ax, and N.L. First (1983) Effects Jalkanen, J., 0. Ritvos, I. Huhtaniemi, U.-H. Stenman, T. Laatikainen, and T. Ranta (1987) Phorbol ester stimulates of cAMP on bovine oocyte maturation and cumulus expanhuman granulosa-luteal cell cyclic adenosine 3'3'-monosion. In: Factors Regulating Ovarian Function. G.S. Greenphosphate and progesterone production. Mol. Cell. Endocriwald and P.F. Terranova, eds. Raven Press, New York, pp. nol., 51:273-276. 61-64. Batta, S.K., and J.F. Knudsen (1980) Calcium concentration Magnusson, C., and T. Hillensjo (1977) Inhibition of maturation and metabolism in rat oocytes by cyclic AMP. J. Exp. in cumulus enclosed oocytes of rats after treatment with ZOO^., 201:139-147. pregnant mare serum. Biol. Reprod., 22:243-246. Berridge, M.J., and R.F. Irvine (1989) Inositol phosphates and Maruska, D.V., M.L. Leibfried, and N.L. First (1984) Role of calcium and the calcium-calmodulin complex in resumption cell signalling. Nature, 341 :197-205. of meiosis, cumulus expansion, viability and hyaluronidase Bornslaeger, E.A., W.T. Poueymirou, P. Mattei, and R.M. sensitivity of bovine cumulus-oocyte complexes. Biol. ReSchultz (1986) Effects of protein kinase C activators on gerprod., 31:l-6. minal vesicle breakdown and polar body emission of mouse McGaughey, R.W., and C. Polge (1971) Cytogenetic analysis oocytes. Exp. Cell Res., 165x507-517. of pig oocytes matured in uitro. J. Exp. Zool., 175:383-396. Budnik, L.T., and A.K. Mukhopadhyay (1987) Desensitization of LH-stimulated cyclic AMP accumulation in isolated McGaughey, R.W., D.H. Montgomery, and J.D. Richter (1979) Germinal vesicle configurations and patterns of polypeptide bovine luteal cells-effect of phorbol ester. Mol. Cell. Ensynthesis of porcine oocytes from antral follicles of different docrinol., 54.51-61. size, as related to their competency for spontaneous maturaCho, W.K., S. Stern, and J.D. Biggers (1974) Inhibitory effect tion. J . Exp. Zool., 209:239-254. of dibutyryl cAMP on mouse oocyte maturation in vivo. J . Michell, R.H. (1986) Inositol phosphates: Profusion and conEXP. ZOO^., 187:383-386. fusion. Nature, 319: 176- 177. Davis, J.S., and M.R. Clark (1983) Activation of protein kinase in the bovine corpus luteum by phospholipid and Ca2+. Nishizuka, Y. (1988)The molecular heterogeneity of protein kinase C and its implications for cellular regulation. NaBiochem. J., 214.569-574. ture, 334:661-665. Davis, J.S., L.L. Weakland, L.A. West, and R.V. Farese
NEOMYCIN AND MEIOSIS Noland, T.A., and M.J. Dimino (1986) Characterization and distribution of protein kinase C in ovarian tissue. Biol. Reprod., 352363472, Paleos, G.A., and R.D. Powers, (1981) The effect of calcium on the first meiotic division of the mammalian oocyte. J . Exp. Zool., 217:409-416. Prentki, M., J.T. Deeney, F.M. Matchinsky, and S.K. Joseph (1986) Neomycin: a specific drug to study the inositolphospholipid signalling system? FEBS Lett., 197:285-288. Rscowsky, C. (1986) The releasing action of calcium upon cyclic AMP-dependent meiotic arrest in hamster oocytes. J . Exp. Zool., 239:263-275. Rice, C., and R.W. McGaughey (1981) Effect of testosterone and dibutyryl CAMPon the spontaneous maturation of pig oocytes. J. Reprod. Fertil. 62:245-256. Sadighian, J.J.,W.G. Kearns, B.J. Waddell, and M.J. Dimino (1989) Effects of diacylglycerol and inositol trisphosphate on steroidogenesis by ovarian granulosa from pigs. Biol. Reprod., 40:294-299. Schacht, J. (1978) Purification of polyphosphoinositides by chromatography on immobilized neomycin. J . Lipid Res., 19:1063-1067. Shinohara, O., M. Knecht, and K.J. Catt (1985) Differential actions of phorbol ester and diacylglycerol on inhibition of granulosa cell maturation. Biochem. Biophys. Res. Commun., 133:468-474. Sirard, M.A., H.M. Florman, M.L. Leibfried-Rutledge, F.L. Barnes, M.L. Sims, and N.L. First (1989) Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes. Biol. Reprod., 40:1257-1263. Stern, S., A. Rayyis, and J.F. Kennedy (1972) Incorporation of amino acids during maturation in vitro by the mouse oocyte:
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effect of puromycin on protein synthesis. Biol. Reprod., 7:341-346. Tsafriri, A. (1978) Oocyte maturation in mammals. In: The Vertebrate Ovary. R.E. Jones, ed. Plenum Press, New York, pp. 409-442. Tysnes, 0.-B., V.M. Steen, and € Holmsen I. (1988) Neomycin inhibits platelet functions and inositol phospholipid metabolism upon stimulation with thrombin, but not with ionomycin or 12-0-tetradecanoyl-phorbol 13-acetate. Eur. J. Biochem., 177:219-223. Umer, F., and S. Schorderet-Slatkine (1984) Inhibition of denuded mouse oocyte meiotic maturation by tumorpromoting phorbol esters and its reversal by retinoids. Exp. Cell Res., 154:600-605. Veldhuis, J.D., and L.M. Demers (1986) An inhibitory role for the protein kinase C pathway in ovarian steroidogenesis. Studies with cultured swine granulosa cells. Biochem. J . 236.505-511. Wasserman, P.M., W.J. Josefowicz, and G.E. Letourneau (1976) Meiotic maturation of mouse oocytes in uitro. Inhibition of maturation at specific stages of nuclear progression. J. Cell Sci., 22.531-545 Wheeler, M.B., and J.D. Veldhuis (1989) Purification of three forms of chromatographically distinct protein kinase C from the swine ovary. Mol. Cell. Endocrinol., 61:117-122. Whitaker, M. (1989) Phosphoinositide second messengers in eggs and oocytes. In: Inositol Lipids in Cell Signalling. R.H. Michell, A.H. Drummond, and C.P. Downes, eds. Academic Press, New York, pp. 459-483. Williamson, J.R., and J.R. Monck (1989) Hormone effects on cellular Ca2+ fluxes. Annu. Rev. Physiol., 51:107-124.
Neomycin, an Inhibitor of Phosphoinositide Hydrolysis, Inhibits the Resumption of Bovine Oocyte Spontaneous Meiotic Maturation SHERYL T. HOMA Department of Zoology, Arizona State University, Tempe, Arizona 85287 The possibility that the intracellular signals generated upon phosphoinositide ABSTRACT hydrolysis are involved in regulating bovine oocyte spontaneous meiotic resumption was investigated. Oocytes were mass-harvested and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin in the presence or absence of neomycin (an inhibitor of phosphoinositide hydrolysis) or phorbol myristate acetate (an activator of protein kinase C). The role of intracellular calcium was examined by preloading with BAPTAIAM (a calcium chelator) prior to culture. Meiotic maturation was scored cytogenetically. 1)Neomycin induces an irreversible inhibition of germinal vesicle breakdown which does not exceed 60% and is apparent at concentrations of 5 mM or above. Progression of meiosis past metaphase I is inhibited at concentrations of 2.5 mM or above. The full effect of neomycin is only apparent if it is presented to the oocytes within 3 h of follicular release, although germinal vesicle breakdown is not observed until 9 h culture under control conditions. 2) PMA alone has negligible effect on germinal vesicle breakdown, but it acts synergistically with 2 mM IBMX to inhibit this process. PMA has a dual effect on the progression of meiosis past metaphase I: 1nM PMA has a stimulatory effect while 1 pM PMA blocks the ability of oocytes to reach anaphase I or beyond. These observations are not found with a non-tumor-promoting phorbol ester. 3) Spontaneous meiotic resumption is not significantly affected in the absence of added exogenous calcium. However, oocytes preloaded with BAPTA/AM exhibit a dose-dependent inhibition of germinal vesicle breakdown, even in the presence of extracellular calcium. These results provide indirect evidence for a role for phosphoinositide metabolism in bovine oocyte maturation; it is suggested that intracellular calcium mobilization is prerequisite for meiotic resumption and protein kinase C may modulate CAMP-dependent inhibition of this response.
Resumption of meiotic maturation in mammalian oocytes is triggered by luteinizing hormone in vivo (Tsafriri, '78). However, the biochemical mechanisms which mediate this process have yet t o be clearly defined. If oocytes are removed from their follicular environment, they will spontaneously mature through all stages of meiosis in culture under defined conditions (Tsafriri, '78). It has been clearly established that elevations in cyclic adenosine monophosphate (CAMP) can inhibit spontaneous meiotic maturation in several mammalian species (Cho et al., '74; Wasserman et al., '76; Magnusson and Hillensjo, '77; Dekel and Beers, '78; Rice and McGaughey, '81; Ball et al., '83; Racowsky, '86; Homa, '881, while increasing evidence supports a central role for calcium in both the resumption and progression of meiosis (Batta and Knudsen, '80; Paleos and Powers, '81; Jagiello et al., '82; Maruska et al., '84; Bae and Channing, '85; Racowsky, '86).It has been demonstrated that calcium levels in intact rat oocytes increase concomitantly with increases in serum0 1991 WILEY-LISS, INC.
luteinizing hormone (Batta and Knudsen, '80) and therefore prior t o initiation of meiotic maturation. In addition, studies on hamster oocytes have revealed that calcium can override meiotic arrest maintained by the cAMP analogue dibutyryl cAMP (Racowsky, '86). It is widely accepted that stimulus-receptoractivated responses which are accompanied by changes in calcium are initiated by membrane phosphoinositide hydrolysis (Michell, '86; Berridge and Irvine, '89; Williamson and Monck, '89). This results in the production of two intermediates: diacylglycerol, which activates protein kinase C (Nishizuka, 'SS), and inositol trisphosphate, which interacts with receptors on the endoplasmic reticulum resulting in mobilization of calcium from within these stores (Berridge and Irvine, '89). While it is known that luteinizing hormone stimulation results in elevated levels of Received March 2, 1990; revision accepted August 2, 1990.
96
S.T. HOMA
CAMP,more recently it has been demonstrated that luteinizing hormone may also activate phosphoinositide turnover in ovarian cells, resulting in an elevation of inositol trisphosphate (Davis et al., '86, '87; Dimino et al., '87; Sadighian et al., '89). Furthermore, protein kinase C, which has been shown to affect CAMP formation in rat (Shinohara et al., '85) and human (Jalkanen et al., '87) granulosa cells, has been characterised in ovarian tissue in both the bovine corpus luteum (Davis and Clark, '83; Noland and Dimino, '86) and in porcine granulosa (Veldhuis and Demers, '86) and luteal (Wheeler and Veldhuis, '89) cells. From the evidence presented above, it appears that phosphoinositide metabolism may play a significant role in mediating the resumption of mammalian oocyte maturation. Neomycin, a polycationic aminoglycoside antibiotic, binds with high affinity to phosphatidylinositol 4,5bisphosphate (Schacht, '78; Gabev et al., '891, thereby interfering with its subsequent hydrolysis and generation of second messengers (Downes and Michell, '81; Tysnes et al., '88; Whitaker, '89). In this study, bovine oocyte spontaneous maturation was examined in the presence of neomycin. In addition, the role of protein kinase C and calcium in meiotic resumption was also investigated. The former was studied by using phorbol myristate acetate (PMA), which mimics the action of diacylglycerol on protein kinase C activity, while BAPTA/AM, a potent calcium chelator which can be trapped inside the cell following deesterification of the acetoxymethyl ester (AM), was used to investigate the latter.
cal Co., St. Louis, MO) were prepared in 2ABMOC medium containing 0.5% bovine serum albumin (fraction V, Sigma Chemical Co.). Stock solutions of phorbol 12-myristate 13-acetate (PMA) (1mM) and 4a-phorbol 12,13-didecanoate (1 mM) (Sigma Chemical Co.) and BAPTA/AM (10 mM) (Calbiochem., La Jolla, CA) were prepared in DMSO such that addition t o the culture medium resulted in a final concentration of 0.2% DMSO. In experiments in which phorbol esters or BAPTA/AM treatment was examined, an equivalent volume of DMSO was added to control cells. For treatment with neomycin, cycloheximide, IBMX, or phorbol esters, oocytes were thoroughly washed in their respective culture medium prior t o incubation. For treatment with BAPTA/AM, groups of 24-36 oocytes were collected in 2ABMOC medium, washed briefly in 150 mM NaCl + 25 mM HEPES, pH 7.2, and then incubated in 2 ml of this medium for 30 min in the presence or absence of BAPTA/AM at 38°C. The loaded oocytes were washed 3 times in 2 ml 2A-BMOC medium containing 0.5%bovine serum albumin t o remove exogenous BAPTA/AM, and then resuspended in 2A-BMOC for long-term culture. Oocytes were cultured in 0.2 ml sterile 2A-BMOC medium, sometimes in the presence of 0.5% bovine serum albumin, and were incubated in wells of sterile Lab-Tek tissue culture chamber slides (#4838, Miles Scientific, Naperville, IL) at 38°C in a humidified atmosphere of 5% C02 + 5% O2 + 90% Nz. Groups of cells (8-12) were treated in duplicate or triplicate for each experiment and each experiment was repeated at least 3 times.
METHODS Collection of oocytes Reproductive tracts from pregnant and nonpregnant cows were obtained from a local slaughterhouse. Ovaries were removed immediately following slaughter. Oocytes devoid of surrounding cumulus cells were purified in chemically defined culture medium, 2A-BMOC, by using a mass harvesting procedure described previously (Homa, '88). Oocytes were then selected for culture with the aid of a light microscope by using established criteria (Home, '88). The average collection time of oocytes from the midpoint of follicular release (0 h) to the start of culture was 1 h.
Determination of meiotic stage Oocytes were fixed, air-dried, and stained (McGaughey and Polge, '71). Oocyte maturation was scored cytogenetically according to criteria previously described (McGaughey et al., '79; Homa, '88). Statistical analyses were carried out by using Student's 't' test.
Culture conditions Sterile solutions of neomycin sulphate (0-10 mM), cycloheximide (10 pg/ml), and 3-isobutyl-lmethyl xanthine (IBMX) (2 mM) (Sigma Chemi-
Protein synthesis Oocytes were cultured in 0.2 ml 2A-BMOC medium containing 0.5% bovine serum albumin, in the presence or absence of either 5 mM neomycin or 10 pg/ml cycloheximide, with 2.5 pCi/ml [U14Clglycine (96 mCi/mmole, New England Nuclear). Three replicates were prepared for each treatment group, containing 20 oocytes per replicate. Incubations were initiated within 3 h of follicular release and terminated after 10 h following follicular release. Incorporation of radio-
NEOMYCIN AND MEIOSIS c
100
-
- %-0 - . tl
,3
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101
0 0
1
2
3
4
5
-
Lo--, 7
6
-8
gE co 0-
oz
9
Neornycin (mM)
Fig. 1. Dose response of meiotic maturation to neomycin. Bovine oocytes were cultured for a total period of 24 h following release from the ovary. Each value represents the mean of 5-12 replicate determinations t S.E.M.
activity into TCA-soluble and -insoluble material was determined as described by Stern et al. ('72).
RESULTS Effect of neomycin on spontaneous meiotic maturation About 90% bovine oocytes devoid of surrounding cumulus cells will spontaneously mature following 24 h in culture as shown in Figure 1. Sixty-nine percent of these maturing oocytes have progressed past metaphase I at this time. In the presence of 5 mM neomycin, there is a 47% inhibition of germinal vesicle breakdown (untreated vs. 5 mM neomycin-treated P < 0.001). This level of inhibition is maintained up to 60% at higher doses of neomycin. At lower concentrations which do not inhibit germinal vesicle breakdown, the proportion of oocytes reaching anaphase I or beyond is significantly reduced (untreated vs. 2.5
97
mM neomycin-treated % anaphase I to metaphase I1 P < O.OOl), while at concentrations of 5 mM or greater, progression of meiosis past metaphase I is almost totally prevented. As neomycin was presented t o the oocytes in the form of a sulphate complex, a dose response t o sodium sulphate up t o 30 mM concentration was carried out. The experiments revealed that sodium sulphate had no significant effect on meitoic maturation (data not shown), suggesting that the effects observed in the presence of neomycin sulphate are attributed t o neomycin. It could be argued that the effects of neomycin on bovine oocyie maturation may be due to the ability of the antibiotic t o inhibit protein synthesis. In the absence of any treatment, germinal vesicle breakdown has occurred in the majority of cultured bovine oocytes by 10 h following release from the ovary (Homa, '88; Sirard et al., '89). 00cytes cultured with as low a concentration as 10 pg/ml cycloheximide for the same period of time demonstrate a significant inhibition of protein synthesis as shown in Table 1(control vs. treated; P < 0.001). Furthermore, 10 pg/ml cycloheximide blocks germinal vesicle breakdown as well as subsequent steps during meiotic maturation of bovine oocytes (Sirard et al., '89). While incubation with 5 mM neomycin causes a significant inhibition of germinal vesicle breakdown (Fig. l),in contrast t o cycloheximide, it failed to inhibit protein synthesis under identical culture conditions (Table 1).Thus the inhibitory effects of neomycin on germinal vesicle breakdown are unlikely to be due to protein synthesis inhibition. The effect of neomycin on germinal vesicle breakdown is not reversible at any dose examined (Fig. 21, as washing oocytes pretreated for 24 h with neomycin and then culturing for an addi-
TABLE 1. Effect of cycloheximide and neomycin on incorporation of [U-I4 Clglycine into oocyte proteins'
Contro1 TCA insoluble (cpm/lO oocytes) TCA soluble ( c p d l 0 oocytes) % TCA insoluble of total uptake % [U-'*C]glycine uptake from medium
*
Cycloheximide (10 pgiml)
* 18"
316
?
96
237
1866
?
181
461 t 35*
170
2184 t 135
2387
17.43 t 0.46**
6.66 t 0.41**
0.658
k
0.032
Neomycin (5 mM)
?
0.621 t 0.051
14.09 t 2.85 0.554 t 0.064
'Values are the mean S.E.M. of three replicate samples. Numbers with the same superscript are significantly different. *, ** P < 0.001.
S.T. HOMA
98 100
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g;
w
201
0-
10
c o
0
0%
0
1
2
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6
7
8
9
10
Nd
Neomycin (mu)
Fig. 2. Reversibility of neomycin effect. Oocytes were cultured for 24 h in increasing concentrations of neomycin, subsequently washed 3 times, and then incubated for a n additional 24 h in the absence of neomycin. Values are the mean of 6-12 replicate determinations t S.E.M.
w 0
2
4
6
8
10 12 14 16 18 20 22 24
Time in control medium (h)
Fig. 3. Determination of the period of sensitivity to neomycin. Bovine oocytes were removed from ovaries at 0 h and cultured in control medium. At intervals of time, groups of 30 oocytes were resuspended in 5 mM neomycin. Incubations were continued for a total of 24 h. Each value represents the mean of 9-13 replicate determinations 2 S.E.M.
tional24 h in the absence of neomycin results in a and an elevation in intracellular levels of calsimilar dose response pattern as treatment with cium, it appeared necessary t o investigate which neomycin for 24 h only. To determine whether of these pathways could influence meiosis. Geroocytes became insensitive t o neomycin treat- minal vesicle breakdown is unaffected by increasment after a period of time in culture, they were ing doses of PMA, which activates protein kinase incubated in control medium for increasing pe- C; however, it has a dual effect on the progression riods of time and then transferred to medium con- of meiosis past metaphase I (Fig. 4a); 1nM PMA taining 5 mM neomycin for a total incubation augments the proportion of oocytes reaching time of 24 h. Figure 3 shows that for over 2 h anaphase I to metaphase I1 (untreated vs. 1 nM following follicular release, oocytes are sensitive PMA-treated P < 0.05) while higher concentrato neomycin treatment, as only 32.4% have tions of the phorbol ester significantly inhibit the undergone germinal vesicle breakdown by the ability of oocytes t o progress beyond metaphase I end of the culture period. However, the ability of 5 (untreated vs. 1 pM PMA-treated P < 0.001). Interestingly, the phorbol ester exerts a dosemM neomycin to inhibit germinal vesicle breakdown is significantly reduced (P < 0.001) by al- dependent synergistic effect on inhibition of germost 50% if it is added at 4 h after release from minal vesicle breakdown in the presence of the follicle. There is a 2 h lag period between 4 IBMX, an inhibitor of CAMP phosphodiesterase and 6 h after follicular release, until the remain- (Fig. 4b). In this study it can be seen that oocytes ing oocytes become insensitive to neomycin. Inhi- will mature in the presence of PMA or 2 mM bition of germinal vesicle breakdown by 5 mM IBMX alone, even though the proportion of matneomycin is abolished if neomycin is presented to uration is significantly decreased in the presence the oocytes at 7 h or more following follicular re- of 2 mM IBMX compared with untreated controls lease. It should be noted that it is not until 9 h (P < 0.02) as previously reported (Homa, '88). following follicular release that bovine oocytes ac- However, in the combined presence of 2 mM tually demonstrate germinal vesicle breakdown IBMX and PMA, the rate of germinal vesicle under control culture conditions in 2A-BMOC breakdown significantly declines at the highest medium in the absence of neomycin (Homa, '88). dose of PMA tested (2 mM IBMX vs. 2 mM IBMX + 1 pM PMA, P < 0.001). IBMX almost comPhorbol ester and spontaneous maturation pletely inhibits the progression of meiosis past One interpretation for the observation that metaphase I and this effect is unaltered in the neomycin inhibits meiotic resumption could im- presence of PMA. The specificity of the effects of PMA was invesplicate a role for phosphoinositide turnover in this process. As hydrolysis of phosphoinositides re- tigated by using similar concentrations of the sults ultimately in activation of protein kinase C non-tumor-promoting phorbol ester, 4a-phorbol
99
NEOMYCIN AND MEIOSIS
a
n
a
. 0
1
0.1
10
100
1000
” Y
Phorbol ester
(nu)
b
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Q
20 10
1
T-
O0I 90
-
-
O
t
0
h
-- -A. 6
S 0.1
1
Y
c.
Y rn
10
100
1000
Phorbol ester
(nu)
Fig. 4. Dose response of meiotic maturation to PMA. 00cytes were incubated with increasing concentrations of PMA (circles) or 4a-phorbol 12J3-didecanoate (triangles) a) in the absence of b) in the presence of 2 mM IBMX for 24 h. Values represent the mean of4-9 replicate determinations 2 S.E.M.
12,13-didecanoate.Figure 4 shows that this phorbol ester has no significant effect either on germinal vesicle breakdown or on the progression of meiosis. Furthermore, unlike PMA, this phorbol ester is inactive in the presence of IBMX.
Calcium and spontaneous maturation The effects of changes in intracellular calcium levels on bovine oocyte meiotic maturation were monitored indirectly by using the calcium chelator BAPTA/AM. The calcium probe was trapped inside oocytes within 90 min of release from the follicle. The oocytes were subsequently washed and incubation was continued in fresh control medium. Figure 5 shows that oocytes preloaded with BAPTA/AM remain at the germinal vesicle stage following 12 h incubation whereas
Fig. 5. Dose response of germinal vesicle breakdown to BAPTMAM treatment. Oocytes were loaded with BAF’TAi AM 1 h following follicular release, then cultured until 12 h following follicular release. Each value represents the mean of 3-8 replicate determinations 2 S.E.M.
untreated cells have reached diakinesis at this time. This inhibition of meiotic resumption is dose dependent, and is evident at concentrations of 10 p M BAPTAiAM or higher (0 vs. 10 p M ,P < 0.02; 0 vs. 20 pM, P < 0.001). These effects are unlikely due to the accumulation of potentially toxic acetoxymethyl esters as the inhibition induced by BAPTA/AM is transient. Table 2 shows that following 24 h culture, the effect of BAPTA/ AM is no longer apparent. It is possible that the BAPTA within the cell becomes saturated with calcium with the constant exchange from the extracellular environment after a period of time, thereby allowing levels of intracellular calcium t o become elevated. By re-treating the oocytes with BAPTA/AM for a second time after 12 h incubation (Table 21, inhibition of meiotic resumption is maintained following 24 h culture (treated vs. untreated P < O.OOl), indicating that prevention of germinal vesicle breakdown by inhibition of elevation of intracellular calcium is not a transient effect. To determine whether the effect of BAPTA is due to inhibition of mobilization of intracellular calcium from intracellular stores, or whether it is due to prevention of calcium movement through the plasma membrane, oocytes preloaded with BAPTA/AM were cultured in the presence of negligible exogenous calcium. Table 3 shows that germinal vesicle breakdown will occur in the absence of added exogenous calcium. Although the proportion of oocytes resuming meiosis under these conditions is reduced from 67.8% in medium routinely supplemented with calcium (1.7 mM) t o
100
S.T. HOMA T A B L E 2. Long-term effects of BAPTAIAM treatment' Experiment A B
Treatment Control 20 (LMBAPTMAM Control 20 LLMBAPTMAM
% Germinal vesicle breakdown
80.4 ? 95.8 ? 96.7 T+_ 15.9 &
16.0 (3) 5.1 (3) 3.6 (6)* 5.5 16)"
'In experiment A, oocytes were treated with or without BAPTAiAM 1 h following follicular release, while in experiment B, oocytes were treated at 1 h and a t 12 h following follicular release. Oocytes were cultured for a total time of 24 h. Values represent the mean i- SEM. Numbers in parentheses represent numbers of replicates. *Significantly different, at P < 0.001.
T A B L E 3. Effect of exogenous calcium on meiotic maturation of oocytes pretreated with or without 20 p,M BAPTAIAM'
peting with calcium for channel binding (Hughes et al., '88; Dulon et al., '89). In addition, it can affect calcium mobilization by chelating inositol % Germinal Vesicle Breakdown trisphosphate in permeabilised cells (Prentki et of Total Oocytes al., '86). However, the effect of neomycin on oocyte CaCb [FMI Untreated Treated maturation is unlikely attributed to its properties 0 45.8 ? 7.2 (7) 7.9 ? 6.5 (7) as a calcium antagonist. Firstly, removal of ex1 45.3 k 4.3 (7) 5.9 ? 4.5 (7) tracellular calcium had no effect on bovine oocyte 10 42.6 ? 8.3 (7) 5.2 ? 4.0 (7) maturation. Secondly, aminoglycoside antibiotics 100 64.9 ? 4.4 (7) 13.2 k 6.7 (7) do not readily penetrate cell membranes (Dulon 1,700 67.8 k 10.0 (6) 4.5 ? 3.1 (6) et al., '89). Even if neomycin did enter the cell in 10ocytes were cultured for 12 h following follicular release. Numbers significant quantities, it is unable to inhibit gerin parentheses represent numbers of experiments. Values represent minal vesicle breakdown beyond 60% whereas themean 2 S.E.M. treatment with the calcium chelator BAPTA/AM almost totally abolishes germinal vesicle break45.8% in the identical medium without calcium down. supplementation, this difference is not signifiIt is of interest that total inhibition of germinal cant. Moreover, this reduction does not occur in vesicle breakdown is not obtained with neomycin, the presence of minimal calcium (100 pM), 17 whereas in the presence of BAPTA/AM, meiotic times less than that normally used for culture (1.7 resumption is almost completely abolished. It is mM). Germinal vesicle breakdown is almost to- generally accepted that inositol trisphosphate, tally abolished in calcium-free media if the cells generated upon phosphatidylinositol 4,5-bisphosare preloaded with BAPTA/AM (45.8 t 7.2% phate hydrolysis, is responsible for binding t o rewithout BAPTA/AM t o 7.9 k 6.5%with BAPTA/ ceptors on the endoplasmic reticulum, stimulrtAM). ing release of calcium into the cytosol (Berridge and Irvine, '89). However, there is strong eviDISCUSSION dence t o suggest that calcium is additionally seThe results of these studies clearly show that questered in intracellular pools which are inacthe phospholipase C inhibitor, neomycin, inhibits cessible t o inositol trisphosphate (Berridge and bovine oocyte maturation, and that this effect is Irvine, '89). While neomycin could effectively premost likely mediated by decreased intracellular vent mobilisation of calcium from the inositol calcium mobilisation. Furthermore, activation of trisphosphate-sensitive pool by inhibition of phosprotein kinase C may be important in regulating pholipase C (Downes and Michell, '81; Tysnes et CAMP-maintained meiotic arrest. al., '88; Whitaker, '89), it has not been shown to While it has been accepted that neomycin in- affect inositol trisphosphate-insensitive pools. hibits phosphoinositide hydrolysis (Downes and Thus, if germinal vesicle breakdown requires calMichell, '81; Tysnes et al., '88; Whitaker, '89>,this cium mobilisation from both the inositol trisphospolycationic antibiotic can also effectively block phate-sensitive and -insensitive pools, then calcium influx across the plasma membrane via neomycin may not be expected t o completely abolmechanisms unrelated to inhibition of phos- ish germinal vesicle breakdown. phoinositide hydrolysis, probably by directly comThe effect of neomycin is apparent only if treat-
NEOMYCIN AND MEIOSIS
ment is within 3 h following release of the oocyte from the follicle. Subsequent treatment with neomycin is only effective in about 50% of responding oocytes after 4 h and this degree of sensitivity is maintained for a n additional 2 h. By 7 h following ovarian release, the oocytes have become committed to mature, as treatment with neomycin at this time is completely ineffective, even though bovine oocytes do not undergo germinal vesicle breakdown until 9 h following follicular release (Homa, '88). Similar experiments carried out in the presence of IBMX have demonstrated that about 50% of responding bovine oocytes become committed to mature following 7 h follicular release (Homa, '88), this sensitivity again being maintained for an additional 2 h period, until 9 h from follicular release when the effects of IBMX are abolished. Thus, the pattern of commitment to maturation in the presence of IBMX compared with that in the presence of neomycin is almost identical, with the exception of a 3 h time difference. These results suggest that phosphoinositide metabolism is enhanced prior t o the period at which they become insensitive to increases in levels of CAMP. Hence, CAMPdependent inhibition of meiotic resumption may be overcome by an event mediated by a second messenger(s) derived from phosphoinositide metabolism. As phosphoinositide turnover yields several important second messengers, it was of interest t o determine which of these could be involved in meiotic maturation. It is possible that diacylglycerol is involved in triggering germinal vesicle breakdown as phorbol esters have been shown t o stimulate rat oocyte maturation (Aberdam and Dekel, '85). On the other hand, activation of protein kinase C has been shown t o inhibit this process in mouse oocytes (Urner and SchorderetSlatkine, '84; Bornslaeger et al., '86). Although activation of protein kinase C by phorbol ester does not inhibit bovine oocyte germinal vesicle breakdown, even at relatively high concentrations, it does have a significant effect on the progression of meiosis. This is consistent with observations made by Bornslaeger et al. ('86) who found that phorbol esters impair the formation of the meiotic spindle, such that polar body emission is prevented. The significance of the dual effect of this phorbol ester on meiotic progression in the bovine oocyte is currently unknown, although it may relate t o the positive and negative feedback roles associated with protein kinase C subspecies (Nishizuka, '88).
101
It is of interest that PMA, while having no demonstrable effect on meiotic resumption, acts synergistically with IBMX, an inhibitor of cAMP phosphodiesterase, to inhibit bovine oocyte germinal vesicle breakdown. Recently it has been shown that phorbol ester can enhance the response of adenylate cyclase t o luteinizinghormone stimulation in bovine luteal cells (Budnik and Mukhopadhyay, '871, and it is well established that cAMP plays a significant role in maintaining meiotic arrest. Protein kinase C has been extracted from ovarian tissue (Davis and Clark, '83; Noland and Dimino, '86; Wheeler and Veldhuis, '89>,and activation of this enzyme enhances levels of cAMP in human granulosa cells (Jalkanen et al., '87). It has been suggested that activation of protein kinase C may result in phosphorylation of the inhibitory subunit of adenylate cyclase, thereby rendering that part of the enzyme complex inactive (Jakobs et al., '86). In the presence of PMA and IBMX, this would result in an enhanced increase of cAMP compared with levels in the presence of IBMX or PMA alone, thus promoting inhibition of germinal vesicle breakdown. As it is possible that bovine oocytes have a very active cAMP phosphodiesterase (Homa, '88), treatment with PMA alone may be expected t o have little effect on meiotic resumption. Indeed, when bovine oocytes are treated with either forskolin or 8-bromo-CAMPto raise levels of CAMP,the inhibitory effects on maturation are masked following prolonged culture, probably due to activity of such a phosphodiesterase (Homa, '88). Investigation of the effects of calcium on bovine oocyte maturation yielded different results. Studies with the calcium probe BAPTA/AM strongly suggest that an increase in intracellular calcium is required for meiotic resumption. Similar effects using BAPTA/AM have also been observed in the porcine oocyte (M. Kaufman and S. T. Homa, unpublished data). In addition, as a considerable proportion of oocytes are capable of undergoing germinal vesicle breakdown in the presence of negligible levels or no added exogenous calcium, this supports the hypothesis that it is mobilization of calcium from intracellular stores that is prerequisite for this process. Previous studies have revealed an absolute dependency on calcium for germinal vesicle breakdown under strictly defined conditions in hamster oocytes (Racowsky,'86). Furthermore, there is a relatively large increase in calcium levels concomitant with meiotic resumption in rat oocytes (Batta and Knudsen, '80).
102
S.T.HOMA
Although many of the effects induced by luteinizing hormone are maintained by CAMP, it has recently been shown that this hormone can stimulate phosphoinositide metabolism (Davis et al., '86, '87; Dimino et al., ,871, resulting in an increase in inositol phosphates. This effect is completely abolished upon activation of protein kinase C (Davis et al., '89). However, these responses have been associated with granulosa cells and the corpus luteum, and the exact mechanism of luteinizing hormone at the level of the oocyte is largely unknown. It would be of great interest to determine whether the mechanisms which appear to be associated with phosphoinositide metabolism during spontaneous meiotic maturation can be extended to hormone-induced meiotic resumption in vivo.
(1986) Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Biochem. J., 238:597-604. Davis, J.S., L.L. Weakland, R.V. Farese, and L.A. West (1987) Luteininzing hormone increases inositol trisphosphate and cytosolic free Ca2+ in isolated bovine luteal cells. J. Biol. Chem., 262:8515-8521. Davis, J.S., L.L. Weakland, R.G. Coffey, and L.A. West (1989) Acute effects of phorbol esters on receptor-mediated IPS, CAMP,and progesterone levels in rat granulosa cells. Am. J. Physiol., 256:E368-E374. Dekel, N., and W.H. Beers (1978) Rat oocyte maturation in vitro: Relief of cyclic AMP inhibition by gonadotropins. Proc. Natl. Acad. Sci., USA, 75:4369-4373. Dimino, M.J., J. Snitzer, and K.M. Brown (1987) Inositol phosphates accumulation in ovarian granulosa after stimulation by luteinizing hormone. Biol. Reprod., 39:1129-1134. Downes, C.P., and R.H. Michell (1981) The polyphosphoinositide phosphodiesterase of erythrocyte membranes. Biochem. J., f98:133-140. Dulon, D., G. Zajic, J.-M. Aran, and J. Schacht (1989) Aminoglycoside antibiotics impair calcium entry but not ACKNOWLEDGMENTS viability and motility in isolated cochlear outer hair cells. J . Neurosci. Res., 24:338-346. The author would like to thank Timothy Wyant E., J. Kasianowicz, T. Abbott, and S. McLaughlin Gabev, and Robert Russell for excellent technical assis(1989) Binding of neomycin to phosphatidylinositol 43tance and Dr. Douglas Chandler for reading the bisphosphate (PIP,). Biochim. Biophys. Acta, 979:105-112. manuscript. This research was supported by Homa, S.T. (1988) Effects of cyclic AMP on the spontaneous grant No. HD 20884 from the National Institutes meiotic maturation of cumulus-free bovine oocytes cultured in chemically defined medium. J. Exp. Zool., 248:222-231. of Health . Hughes B.P., A.M. Auld, and G.J. Barritt (1988) Evidence that neomycin inhibits plasma membrane Ca2+ inflow in LITERATURE CITED isolated hepatocytes. Biochem. 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