ReproductiveToxicology, Vol. 6, pp. 309-318, 1992
0890-6238/92 $5.00 + ,00 Copyright© 1992 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved,
CAFFEINE
EFFECTS
ON MEIOTIC MATURATION OOCYTES IN VITRO
IN HAMSTER
A N G E L A L. P R A T H E R * a n d C A T H E R I N E R A C O W S K Y t *Department of Zoology, Arizona State University, Tempe, Arizona; i'Department of Obstetrics and Gynecology, University of Arizona Health Sciences Center, Tucson, Arizona Abstract - - The effect of caffeine on meiotic maturation in cultured hamster oocytes was investigated. Meiotic status was scored from chromatin spreads of oocytes previously exposed to caffeine (0, 0.00017, 0.0017, 0.017, 0.17, 1.7, 2.4, 5.1, and 10.2 mM) for up to 20 h. While concentrations of caffeine less than 0.017 mM failed to affect significantly the onset of meiotic resumption, 0.0017 mM caffeine significantly decreased the proportion of oocytes progressing normally to telophase l-metaphase II, and concomitantly increased the proportion of both diploid MII and anenploid oocytes. In addition, 0.17 to 10.2 mM caffeine induced a dose-dependent increase in the proportion of meiotically arrested oocytes, with less than 5% oocytes progressing normally through to the final stages of meiotic maturation at 10.2 mM caffeine. Taken together, these data show that caffeine at concentrations as low as 0.0017 to 0.017 mM interfere with progression of meiotic maturation, and that concentrations higher than 0.017 mM delay initiation of this process. Since caffeine peaks at 0.017 mM in the plasma of women following a cup of brewed coffee, we conclude that caffeine-induced perturbations of oocyte meiotic maturation may be responsible, at least in part, for the recently revealed correlation between caffeine intake and reduced fertility in women. Key Words: caffeine;oocyte; meiotic maturation; hamster; culture.
cyte meiotic maturation. Thus, caffeine causes translocation of Ca 2+ from intracellular storage sites into the cytosol (3), and there is considerable evidence that Ca 2+ plays a central role in the regulation of meiotic maturation in mammalian oocytes (4). In addition, caffeine and its dimethylxanthine metabolites, theophylline and theobromine, are established inhibitors of cyclic adenosine monophosphate (cyclic AMP) phosphodiesterase (5), and cyclic AMP appears to play a key role in the regulation of meiotic maturation in mammalian oocytes (see reference 6 for a review). Taken together, these observations raise the possibility that the reduced fertility of women who consume caffeine may be due to a direct action of the alkaloid on the meiotic process of the oocyte prior to ovulation. The research described in this paper has examined this possibility using the oocyte of the golden Syrian hamster as the model system. Oocytes were cultured in various concentrations of caffeine, and the effects of such exposure on both the resumption and the progression of meiosis were determined.
INTRODUCTION While ingestion of caffeine has been associated with a variety of adverse physiologic conditions, including various reproductive disorders (1), little is known regarding the effects of caffeine and its metabolites on fertility. However, an epidemiologic study has revealed a significant positive correlation between the amount of caffeine consumed by women attempting to conceive and the time taken for conception to occur. This observation, therefore, provides evidence that an association exists between caffeine ingestion and decreased human fecundity (2). The mechanisms that mediate these adverse effects of caffeine on human fertility have not been elucidated. Thus, it is unknown whether caffeine impairs fertility by a direct action on oocyte meiotic progression or on early embryonic development, or whether this alkaloid exerts an indirect effect on the genital tract. In support of a direct effect on the oocyte, however, is the fact that several of the established cellular responses elicited by caffeine and its dimethylxanthine metabolites are believed to be involved in oo-
MATERIALS AND M E T H O D S
Collection of oocytes
Address correspondence to Dr. Catherine Racowsky, Department of Obstetrics & Gynecology, The University of Arizona Health Sciences Center, Tucson, AZ 85724 Received 9 August 1991; Revision Received 16 October 1991; Accepted 20 October 1991.
Oocytes were collected from mature golden Syrian hamsters (Mesocricetus auratus) weighing between 80 and 120 g that were maintained in lighting 309
310
Reproductive Toxicology
conditions of 14L: 10D (lights on at 0500 h) with food and water provided ad libitum. Animals were injected with 30 IU pregnant mare's serum gonadotropin (PMSG) in 0.1 mL saline (Sigma, St. Louis, MO) at metestrus between 0800 and 1100 h as previously described (7), after exhibiting the vaginal discharge characteristic of metestrus for at least two consecutive estrous cycles. The animals were asphyxiated with CO2 48 h after the PMSG injection. The ovaries were removed immediately, and each pair of ovaries was placed in a sterile Falcon petri dish # 1008 containing 2 mL of 3BSA-BMOC (8). The largest follicles ( > 0.5-mm diameter) were punctured with a sterile 26-gauge needle attached to a sterile 3-mL syringe into 3BSA-BMOC medium under a dissecting microscope. Only those oocytes with a tightly adherent, intact mass of cumulus cells were selected for subsequent experiments, and since the oocytes were collected one day before the beginning of the preovulatory surge of LH (9), none of the cumuli were expanded. In those experiments utilizing cumulusfree oocytes, the cumuli from selected cumulus-enclosed oocytes were mechanically removed with a fine bore pipet (t0). Since 50% of hamster oocytes undergo irreversible commitment to maturation within 1 h of follicular release (4), all oocytes were placed in the appropriate culture medium within 30 min of follicular release.
Culture system Oocytes were cultured in a humidified atmosphere of 5% 02 + 5% CO2 + 90% N2 in modified Medium 199 (TCM, reference 11) in a system identical to that previously described for rat oocytes (12). On the morning of each experiment, fresh TCM and a stock solution of caffeine were prepared. The concentration of this stock was equal to that of the highest concentration of caffeine to be tested; all other test media were prepared from this stock by serial dilution. All media were sterilized through a 0.22-um Millipore filter and were equilibrated to culture conditions for at least 1 h before use. Groups of 15 to 20 oocytes (those obtained from one ovary) were cultured as replicates (an average of 7 oocytes per replicate) for 4, 8, or 20 h, as indicated for each series of experiments. Within an experiment, two replicates were normally provided for each treatment, and each experiment was repeated at least three times. After culture, oocytes were prepared for light microscopy (13).
Experimental design Four series of experiments were undertaken in this study. In the first and second series, the effects of caffeine on the resumption and progression of meio-
Volume 6, Number 4, 1992
sis were investigated by culturing cumulus-enclosed oocytes for 4 and 20 h, respectively, in the presence of various concentrations of caffeine (0, 0.00017, 0.0017, 0.017, 0.17, 1.7, 2.4, 5.1, and 10.2 raM). Thus, caffeine concentrations were investigated that were less than or greater than 0.017 mM, which is the peak caffeine concentration in the plasma of women following an oral dose of I15 mg (14), the amount of caffeine present in one cup of brewed coffee (15). In the third series, the reversibility of caffeinemaintained meiotic arrest was investigated by comparing the meiotic progression of oocytes exposed to caffeine during an initial culture period with that of control, unexposed oocytes. In the fourth series, the mediating role of the cumulus cells in the maintenance of caffeine-induced meiotic arrest was studied.
Determination of oocyte meiotic stage Where necessary, cumulus cells were removed and the oocytes were air-dried as described previously (13) onto microscope slides in groups corresponding to the wells in which they were cultured. The air-dried preparations were subsequently stained with Wright's stain in 2.9% (w/v) glycerol and 97.1% methanol (16), and the resultant chromatin spreads were scored as a germinal vesicle (GV), early diakinesis, diakinesis, late diakinesis, prometaphase I, metaphase I, anaphase I, telophase I, or metaphase II. Typical configurations of a GV, and a diakinesis, prometaphase I, metaphase I, anaphase I, and metaphase II are shown in Figure 1. Four aberrations in this normal meiotic sequence were observed: 1) an aberration of metaphase I in which the chromosomes were clumped (Figure 2a); 2) diploid metaphase II, in which the homologues failed to segregate at the first meiotic division (Figure 2b); 3) an anomaly in which the bivalents, rather than the homologues, segregated to opposite poles of the meiotic spindle (Figure 2d), resulting in a chromosome distribution designated as two groups of metaphase I chromosomes (Figure 2c); 4) aneuploidy, in which only some of the homologues segregated during anaphase, giving rise to an oocyte with an abnormal number of chromosomes that was either hyperhaploid (Figure 2e and 2f) or hypohaploid (Figure 2g). For each experiment, the proportions of GVstage oocytes, and oocytes at metaphase I, beyond metaphase I and at telophase I-metaphase II were determined. In addition, the number of oocytes with diploid metaphase II chromosomes, and with an aneuploid congiguration were determined and expressed as a percentage of all the oocytes examined within a group.
Caffeineand oocytematuration• A. L. PRATHERand C. RACOWSKY
31 1
Fig. 1. Chromatin configurations during meiotic maturation in hamster oocytes. (a) Germinal vesicle(GV) of an oocyte after 0 h culture, magnified × 708 (arrow indicates uncondensed chromatin); (b) Diakinesis of an oocyte after 4 h of culture, magnified × 775 (arrow indicates condensing and synapsed chromatin); (c) Metaphase I of an oocyte after 4 h of culture, magnified × 1308; (d) Anaphase I of an oocyte after 8 h of culture, magnified × 1417 (arrow indicates position of metaphase plate); (e) Telophase I of an oocyte after 20 h of culture, magnified X 1188; (f) Metaphase II of an oocyte after 20 h of culture, magnified × 1278. The arrows in Panels e and f indicate the polar body chromosomes, which are undergoing progressive degeneration through telophase I to metaphase II.
Statistical analyses Due to heterogeneity of variances for all of the parameters examined, the data were logarithmically transformed prior to analysis, except for those data expressed as proportions, which were transformed to arcsines. An SAS general linear model for analysis of variance for an unbalanced design (PROC GLM, reference 17), followed by Duncan's Multiple Ranges Test for multiple comparisons (18), was used to test for significant (P < 0.05) differences among the various treatment groups. Group means were transformed back to absolute values for graphic presentation or tabulation. Student's paired t test was used to assess significant differences when paired comparisons were undertaken, again with P < 0.05 considered statistically significant.
RESULTS The first series of experiments examined the effect of various concentrations of caffeine on meiotic resumption in cumulus-enclosed oocytes following 4 h of culture. Figure 3a shows that a complex doseresponse curve was generated when comparing the proportion ofGV-stage oocytes to the dose of caffeine added to the culture medium. Thus, five phases were revealed, the first of which occurred between 0 and 0.017 mM and was less pronounced than the following phases. The second phase occurred between 0.017 mM and 1.7 mM and comprised a sharp rise, followed by a more shallow response between 0.17 mM and 1.7 mM. The third phase, which occurred between 1.7 mM and 2.4 mM, had a gradient that was
Fig. 2. Caffeine-induced disruption in the oocyte meiotic process. (a) Clumped metaphase I chromosomes from an oocyte following 4-h culture in the presence of 0.0017 mM caffeine, in which the bivalents appear tightly apposed to one another, magnification X t 750; (b) A diploid metaphase 1I configuration of an oocyte cultured for 20 h in 0.17 mM caffeine, in which the homologues failed to segregate, giving rise to a diploid chromosomal complement, magnified X 1t 35; (c) Two groups of metaphase I chromosomes from an oocyte cuRured for 20 h in 0.17 mM caffeine, in which the bivalents, rather than the homologues, segregated to the poles, magnified × 1174 (note that one set of bivalents is undergoing degeneration [marked by an arrow], thereby mimicking degeneration of normal polar body chromosomes); (d) An aberrant anaphase I of an oocyte cultured for 20 h in 1.7 mM caffeine, in which segregation of several of the homologues failed (marked by arrow heads), magnified X 1456; the arrow indicates the position of the metaphase plate. (e-g) Aneuploid chromosomal configurations of oocytes in which unequal segregation of homologues occurred at anaphase I, resulting in hyperhaploid (Figure 2e and f) and hypohaploid (Figure 2g) chromosomal complements following 20-h culture in 0.0017 mM, 5.1 mM, and 0. t 7 mM caffeine, respectively, magnified X 1375 (e), X 1331 (f), and X 1013 (g); the arrows indicate polar body chromosomes in Panel e and the oocyte chromosomes in Panels fand g. 312
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Fig. 3. The effectof caffeineon meiotic resumption in hamster oocytes. Chromatin spreads of oocytes, previously cultured for 4 h in medium containing various concentrations of caffeine, were scored for the proportion at the GV stage (a) or with metaphase I chromosomes (b). Each value represents the mean _+ SEM of between 8 and 12 replicates. greater than that of either the first or the second phase, but which was less steep than that of the fourth phase, which occurred between 2.4 mM and 5.1 raM. The final phase, in the caffeine range between 5.1 mM and 10.2 mM, exhibited a less steep slope than that of the two immediately preceding phases. Interestingly, during the first and third phases, a parallelism existed between the proportion of GV-stage oocytes (Figure 3a) and the proportion of maturing oocytes that had reached metaphase I (Figure 3b). In contrast, during phases two and four, opposite trends were revealed. Over the range of caffeine concentrations tested, 2.4 mM caffeine maintained 50% of oocytes at the GVstage. Following exposure for 20 h to the above concentrations of caffeine, a typical sigmoidal dose-response curve was generated (Figure 4a) disclosing that 2.8 mM caffeine inhibited 50% of the oocytes from undergoing GV breakdown. Between 0.017 mM and 1.7 mM, there was a significant reduction in the proportion of GV-stage oocytes at 20 h, as corn-
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Fig. 4. The effectof caffeineon meiotic progression in hamster oocytes. Following 20 h of exposure to various concentrations of caffeine, oocytes were scored cytogeneticallyfor the proportion either at the GV stage (a) or beyond metaphase I (b). Each value represents the mean + SEM of between 8 and 12 replicates. pared to that observed after 4 h of culture (0.17 mM: 30.9 + 12.0 compared with 2.4 + 2.4 at 4 and 20 h, respectively, P < 0.000 I, Student's paired t test; 1.7 mM: 56.1 + 10.0 compared with 9.0 _+ 4.5 at 4 and 20 h, respectively, P < 0.0001, Student's paired t test). In addition, the average combined proportion ofoocytes remaining at the GV-stage after 20 h in the presence of 5.1 mM and 10.2 mM caffeine was significantly lower than that observed for the corresponding caffeine doses following 4 h of culture (78.4% compared with 95.3%, respectively, P < 0.0004, Student's paired t test). The data presented in Figure 4b show that while slightly more than 60% ofoocytes progressed beyond metaphase I when cultured under controlled conditions, a very dramatic reduction in this proportion was observed in those groups exposed to caffeine concentrations greater than 2.4 mM. However, a marginal decrease in the proportion of post-metaphase I
314
Reproductive Toxicology
oocytes was revealed in those groups exposed to caffeine ranging between 0.00017 mM and 0.0017 mM, which was followed by an upward trend to 1.7 mM. Further analysis of the oocytes that had progressed beyond metaphase I revealed that they fell into three major groups: 1) those oocytes exhibiting a normal telophase I/metaphase II configuration; 2) those oocytes at diploid metaphase II; and 3) those oocytes that were aneuploid, but that did not exhibit a typical diploid metaphase II configuration (Figure 5). The proportion ofoocytes that contained normal telophase I-metaphase II configurations fluctuated between 0 m M and 0.0017 mM but underwent a progressive, albeit gradual, increase between 0.0017 and 1.7 mM that was followed by a dramatic rise between 1.7 mM and 5.1 mM. This trend was opposite to that observed for the proportion of post-metaphase I oocytes that were at diploid metaphase II; however, while the trend for aneuploid oocytes followed that for diploid metaphase II oocytes up to 0.0017 mM, and paralleled that for the proportion of normal telophase I-metaphase II oocytes between 0.0017 mM and 1.7 mM, it was diametrically opposed to that for normal mature oocytes between 1.7 mM and 10.2 mM. Of all aneuploid oocytes observed (n = 27), 81.48% were hyperhaploid, the incidence of hyperhaploidy being unrelated to the dose of caffeine tested. Since the proportion of meiotically arrested oocytes cultured for 4 h (Figure 3a) followed a trend similar to that for oocytes exhibiting normal telo-
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Volume 6. Number 4, 1992
phase I-metaphase II configurations following 20 h of culture (Figure 5), analyses were undertaken to determine the relevance of an initial period of meiotic arrest in cultured oocytes to completion of normal meiotic maturation. Given the relative lack of effect of caffeine in the maintenance of meiotic arrest at doses up to 0.017 mM, the data were analyzed in two groups: the first including oocytes cultured in the presence of 0 to 0.017 mM caffeine, and the second including oocytes cultured in caffeine at 0.17 to 10.2 mM. As shown in Figure 6, a positive correlation between the % GV and the % normal telophase I-metaphase II was observed for both groups, this relationship being statistically significant for oocytes exposed to the higher caffeine doses (0.17 to 10.2 mM; [r] = 0.987, P < 0.02, where [r] = the correlation coefficient). In contrast, negative correlations were observed between the % GV and the proportion of either diploid metaphase II or aneuploid oocytes, these correlations also being statistically significant only for the higher dose-range of caffeine tested ([ r] = - 0.988, P < 0.002, and [r] = -0.975, P < 0.05, respectively). Since less than 25% ofoocytes had resumed meiosis following 20 h of culture in the presence of 5.1 or 10.2 mM of caffeine (Figure 4), the possibility existed that these high doses of caffeine had an irreversible, and adverse, effect on oocyte meiotic competency. A series of experiments was conducted, therefore, in which the reversibility of caffeine-induced maintenance of meiotic arrest was investigated. Cumulusenclosed oocytes were cultured in the presence of 5.1 mM caffeine for 4 h, after which they were either airdried for analysis or transferred to fresh medium for an additional 4 h culture in the absence (control) or presence of 5.1 mM caffeine. When exposed continually to 5.1 mM caffeine, less than 20% of oocytes resumed meiosis, whether they were cultured for 4 or for 8 h (Table 1). In contrast, those oocytes transferred to control medium exhibited a high incidence of meiotic resumption with only 5% maintained at the GV stage.
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[Coffeine] mM Fig. 5. The effect o f caffeine on perturbations o f the first meiotic division in hamster oocytes. Following 20 h of culture in media containing various concentrations o f caffeine, oocytes were scored cytogenetically for the proportion that exhibited n o r m a l telophase I/metaphase II configurations (open diamonds), diploid metaphase II c h r o m o s o m e s (closed diamonds), or that were aneuploid (open triangles). Each value represents the m e a n + SEM o f 8 to 12 replicates.
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Caffeine and oocyte maturation • A. L. PRATHERand C. RACOWSKY
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containing either 0 m M (control) or 5.1 mM caffeine. As shown in "Fable 2, 0% of oocytes remained at the GV-stage when cultured in the absence of caffeine, regardless of the presence of cumulus cells. In contrast, following exposure to 5.1 mM caffeine, the majority of oocytes, in all three groups, were maintained in meiotic arrest. Interestingly, however, in caffeine-free media, the meiotic stage reached by maturing oocytes was significantly affected by the presence of adherent cumulus cells. Thus, compared with the proportion of maturing oocytes at diakinesis-metaphase I in the cumulus-free group either cultured alone or in the presence of loose cumulus cells, that of cumulus-enclosed oocytes was significantly greater in control medium (Table 2).
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0890-6238/92 $5.00 + ,00 Copyright© 1992 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved,
CAFFEINE
EFFECTS
ON MEIOTIC MATURATION OOCYTES IN VITRO
IN HAMSTER
A N G E L A L. P R A T H E R * a n d C A T H E R I N E R A C O W S K Y t *Department of Zoology, Arizona State University, Tempe, Arizona; i'Department of Obstetrics and Gynecology, University of Arizona Health Sciences Center, Tucson, Arizona Abstract - - The effect of caffeine on meiotic maturation in cultured hamster oocytes was investigated. Meiotic status was scored from chromatin spreads of oocytes previously exposed to caffeine (0, 0.00017, 0.0017, 0.017, 0.17, 1.7, 2.4, 5.1, and 10.2 mM) for up to 20 h. While concentrations of caffeine less than 0.017 mM failed to affect significantly the onset of meiotic resumption, 0.0017 mM caffeine significantly decreased the proportion of oocytes progressing normally to telophase l-metaphase II, and concomitantly increased the proportion of both diploid MII and anenploid oocytes. In addition, 0.17 to 10.2 mM caffeine induced a dose-dependent increase in the proportion of meiotically arrested oocytes, with less than 5% oocytes progressing normally through to the final stages of meiotic maturation at 10.2 mM caffeine. Taken together, these data show that caffeine at concentrations as low as 0.0017 to 0.017 mM interfere with progression of meiotic maturation, and that concentrations higher than 0.017 mM delay initiation of this process. Since caffeine peaks at 0.017 mM in the plasma of women following a cup of brewed coffee, we conclude that caffeine-induced perturbations of oocyte meiotic maturation may be responsible, at least in part, for the recently revealed correlation between caffeine intake and reduced fertility in women. Key Words: caffeine;oocyte; meiotic maturation; hamster; culture.
cyte meiotic maturation. Thus, caffeine causes translocation of Ca 2+ from intracellular storage sites into the cytosol (3), and there is considerable evidence that Ca 2+ plays a central role in the regulation of meiotic maturation in mammalian oocytes (4). In addition, caffeine and its dimethylxanthine metabolites, theophylline and theobromine, are established inhibitors of cyclic adenosine monophosphate (cyclic AMP) phosphodiesterase (5), and cyclic AMP appears to play a key role in the regulation of meiotic maturation in mammalian oocytes (see reference 6 for a review). Taken together, these observations raise the possibility that the reduced fertility of women who consume caffeine may be due to a direct action of the alkaloid on the meiotic process of the oocyte prior to ovulation. The research described in this paper has examined this possibility using the oocyte of the golden Syrian hamster as the model system. Oocytes were cultured in various concentrations of caffeine, and the effects of such exposure on both the resumption and the progression of meiosis were determined.
INTRODUCTION While ingestion of caffeine has been associated with a variety of adverse physiologic conditions, including various reproductive disorders (1), little is known regarding the effects of caffeine and its metabolites on fertility. However, an epidemiologic study has revealed a significant positive correlation between the amount of caffeine consumed by women attempting to conceive and the time taken for conception to occur. This observation, therefore, provides evidence that an association exists between caffeine ingestion and decreased human fecundity (2). The mechanisms that mediate these adverse effects of caffeine on human fertility have not been elucidated. Thus, it is unknown whether caffeine impairs fertility by a direct action on oocyte meiotic progression or on early embryonic development, or whether this alkaloid exerts an indirect effect on the genital tract. In support of a direct effect on the oocyte, however, is the fact that several of the established cellular responses elicited by caffeine and its dimethylxanthine metabolites are believed to be involved in oo-
MATERIALS AND M E T H O D S
Collection of oocytes
Address correspondence to Dr. Catherine Racowsky, Department of Obstetrics & Gynecology, The University of Arizona Health Sciences Center, Tucson, AZ 85724 Received 9 August 1991; Revision Received 16 October 1991; Accepted 20 October 1991.
Oocytes were collected from mature golden Syrian hamsters (Mesocricetus auratus) weighing between 80 and 120 g that were maintained in lighting 309
310
Reproductive Toxicology
conditions of 14L: 10D (lights on at 0500 h) with food and water provided ad libitum. Animals were injected with 30 IU pregnant mare's serum gonadotropin (PMSG) in 0.1 mL saline (Sigma, St. Louis, MO) at metestrus between 0800 and 1100 h as previously described (7), after exhibiting the vaginal discharge characteristic of metestrus for at least two consecutive estrous cycles. The animals were asphyxiated with CO2 48 h after the PMSG injection. The ovaries were removed immediately, and each pair of ovaries was placed in a sterile Falcon petri dish # 1008 containing 2 mL of 3BSA-BMOC (8). The largest follicles ( > 0.5-mm diameter) were punctured with a sterile 26-gauge needle attached to a sterile 3-mL syringe into 3BSA-BMOC medium under a dissecting microscope. Only those oocytes with a tightly adherent, intact mass of cumulus cells were selected for subsequent experiments, and since the oocytes were collected one day before the beginning of the preovulatory surge of LH (9), none of the cumuli were expanded. In those experiments utilizing cumulusfree oocytes, the cumuli from selected cumulus-enclosed oocytes were mechanically removed with a fine bore pipet (t0). Since 50% of hamster oocytes undergo irreversible commitment to maturation within 1 h of follicular release (4), all oocytes were placed in the appropriate culture medium within 30 min of follicular release.
Culture system Oocytes were cultured in a humidified atmosphere of 5% 02 + 5% CO2 + 90% N2 in modified Medium 199 (TCM, reference 11) in a system identical to that previously described for rat oocytes (12). On the morning of each experiment, fresh TCM and a stock solution of caffeine were prepared. The concentration of this stock was equal to that of the highest concentration of caffeine to be tested; all other test media were prepared from this stock by serial dilution. All media were sterilized through a 0.22-um Millipore filter and were equilibrated to culture conditions for at least 1 h before use. Groups of 15 to 20 oocytes (those obtained from one ovary) were cultured as replicates (an average of 7 oocytes per replicate) for 4, 8, or 20 h, as indicated for each series of experiments. Within an experiment, two replicates were normally provided for each treatment, and each experiment was repeated at least three times. After culture, oocytes were prepared for light microscopy (13).
Experimental design Four series of experiments were undertaken in this study. In the first and second series, the effects of caffeine on the resumption and progression of meio-
Volume 6, Number 4, 1992
sis were investigated by culturing cumulus-enclosed oocytes for 4 and 20 h, respectively, in the presence of various concentrations of caffeine (0, 0.00017, 0.0017, 0.017, 0.17, 1.7, 2.4, 5.1, and 10.2 raM). Thus, caffeine concentrations were investigated that were less than or greater than 0.017 mM, which is the peak caffeine concentration in the plasma of women following an oral dose of I15 mg (14), the amount of caffeine present in one cup of brewed coffee (15). In the third series, the reversibility of caffeinemaintained meiotic arrest was investigated by comparing the meiotic progression of oocytes exposed to caffeine during an initial culture period with that of control, unexposed oocytes. In the fourth series, the mediating role of the cumulus cells in the maintenance of caffeine-induced meiotic arrest was studied.
Determination of oocyte meiotic stage Where necessary, cumulus cells were removed and the oocytes were air-dried as described previously (13) onto microscope slides in groups corresponding to the wells in which they were cultured. The air-dried preparations were subsequently stained with Wright's stain in 2.9% (w/v) glycerol and 97.1% methanol (16), and the resultant chromatin spreads were scored as a germinal vesicle (GV), early diakinesis, diakinesis, late diakinesis, prometaphase I, metaphase I, anaphase I, telophase I, or metaphase II. Typical configurations of a GV, and a diakinesis, prometaphase I, metaphase I, anaphase I, and metaphase II are shown in Figure 1. Four aberrations in this normal meiotic sequence were observed: 1) an aberration of metaphase I in which the chromosomes were clumped (Figure 2a); 2) diploid metaphase II, in which the homologues failed to segregate at the first meiotic division (Figure 2b); 3) an anomaly in which the bivalents, rather than the homologues, segregated to opposite poles of the meiotic spindle (Figure 2d), resulting in a chromosome distribution designated as two groups of metaphase I chromosomes (Figure 2c); 4) aneuploidy, in which only some of the homologues segregated during anaphase, giving rise to an oocyte with an abnormal number of chromosomes that was either hyperhaploid (Figure 2e and 2f) or hypohaploid (Figure 2g). For each experiment, the proportions of GVstage oocytes, and oocytes at metaphase I, beyond metaphase I and at telophase I-metaphase II were determined. In addition, the number of oocytes with diploid metaphase II chromosomes, and with an aneuploid congiguration were determined and expressed as a percentage of all the oocytes examined within a group.
Caffeineand oocytematuration• A. L. PRATHERand C. RACOWSKY
31 1
Fig. 1. Chromatin configurations during meiotic maturation in hamster oocytes. (a) Germinal vesicle(GV) of an oocyte after 0 h culture, magnified × 708 (arrow indicates uncondensed chromatin); (b) Diakinesis of an oocyte after 4 h of culture, magnified × 775 (arrow indicates condensing and synapsed chromatin); (c) Metaphase I of an oocyte after 4 h of culture, magnified × 1308; (d) Anaphase I of an oocyte after 8 h of culture, magnified × 1417 (arrow indicates position of metaphase plate); (e) Telophase I of an oocyte after 20 h of culture, magnified X 1188; (f) Metaphase II of an oocyte after 20 h of culture, magnified × 1278. The arrows in Panels e and f indicate the polar body chromosomes, which are undergoing progressive degeneration through telophase I to metaphase II.
Statistical analyses Due to heterogeneity of variances for all of the parameters examined, the data were logarithmically transformed prior to analysis, except for those data expressed as proportions, which were transformed to arcsines. An SAS general linear model for analysis of variance for an unbalanced design (PROC GLM, reference 17), followed by Duncan's Multiple Ranges Test for multiple comparisons (18), was used to test for significant (P < 0.05) differences among the various treatment groups. Group means were transformed back to absolute values for graphic presentation or tabulation. Student's paired t test was used to assess significant differences when paired comparisons were undertaken, again with P < 0.05 considered statistically significant.
RESULTS The first series of experiments examined the effect of various concentrations of caffeine on meiotic resumption in cumulus-enclosed oocytes following 4 h of culture. Figure 3a shows that a complex doseresponse curve was generated when comparing the proportion ofGV-stage oocytes to the dose of caffeine added to the culture medium. Thus, five phases were revealed, the first of which occurred between 0 and 0.017 mM and was less pronounced than the following phases. The second phase occurred between 0.017 mM and 1.7 mM and comprised a sharp rise, followed by a more shallow response between 0.17 mM and 1.7 mM. The third phase, which occurred between 1.7 mM and 2.4 mM, had a gradient that was
Fig. 2. Caffeine-induced disruption in the oocyte meiotic process. (a) Clumped metaphase I chromosomes from an oocyte following 4-h culture in the presence of 0.0017 mM caffeine, in which the bivalents appear tightly apposed to one another, magnification X t 750; (b) A diploid metaphase 1I configuration of an oocyte cultured for 20 h in 0.17 mM caffeine, in which the homologues failed to segregate, giving rise to a diploid chromosomal complement, magnified X 1t 35; (c) Two groups of metaphase I chromosomes from an oocyte cuRured for 20 h in 0.17 mM caffeine, in which the bivalents, rather than the homologues, segregated to the poles, magnified × 1174 (note that one set of bivalents is undergoing degeneration [marked by an arrow], thereby mimicking degeneration of normal polar body chromosomes); (d) An aberrant anaphase I of an oocyte cultured for 20 h in 1.7 mM caffeine, in which segregation of several of the homologues failed (marked by arrow heads), magnified X 1456; the arrow indicates the position of the metaphase plate. (e-g) Aneuploid chromosomal configurations of oocytes in which unequal segregation of homologues occurred at anaphase I, resulting in hyperhaploid (Figure 2e and f) and hypohaploid (Figure 2g) chromosomal complements following 20-h culture in 0.0017 mM, 5.1 mM, and 0. t 7 mM caffeine, respectively, magnified X 1375 (e), X 1331 (f), and X 1013 (g); the arrows indicate polar body chromosomes in Panel e and the oocyte chromosomes in Panels fand g. 312
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Fig. 3. The effectof caffeineon meiotic resumption in hamster oocytes. Chromatin spreads of oocytes, previously cultured for 4 h in medium containing various concentrations of caffeine, were scored for the proportion at the GV stage (a) or with metaphase I chromosomes (b). Each value represents the mean _+ SEM of between 8 and 12 replicates. greater than that of either the first or the second phase, but which was less steep than that of the fourth phase, which occurred between 2.4 mM and 5.1 raM. The final phase, in the caffeine range between 5.1 mM and 10.2 mM, exhibited a less steep slope than that of the two immediately preceding phases. Interestingly, during the first and third phases, a parallelism existed between the proportion of GV-stage oocytes (Figure 3a) and the proportion of maturing oocytes that had reached metaphase I (Figure 3b). In contrast, during phases two and four, opposite trends were revealed. Over the range of caffeine concentrations tested, 2.4 mM caffeine maintained 50% of oocytes at the GVstage. Following exposure for 20 h to the above concentrations of caffeine, a typical sigmoidal dose-response curve was generated (Figure 4a) disclosing that 2.8 mM caffeine inhibited 50% of the oocytes from undergoing GV breakdown. Between 0.017 mM and 1.7 mM, there was a significant reduction in the proportion of GV-stage oocytes at 20 h, as corn-
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Fig. 4. The effectof caffeineon meiotic progression in hamster oocytes. Following 20 h of exposure to various concentrations of caffeine, oocytes were scored cytogeneticallyfor the proportion either at the GV stage (a) or beyond metaphase I (b). Each value represents the mean + SEM of between 8 and 12 replicates. pared to that observed after 4 h of culture (0.17 mM: 30.9 + 12.0 compared with 2.4 + 2.4 at 4 and 20 h, respectively, P < 0.000 I, Student's paired t test; 1.7 mM: 56.1 + 10.0 compared with 9.0 _+ 4.5 at 4 and 20 h, respectively, P < 0.0001, Student's paired t test). In addition, the average combined proportion ofoocytes remaining at the GV-stage after 20 h in the presence of 5.1 mM and 10.2 mM caffeine was significantly lower than that observed for the corresponding caffeine doses following 4 h of culture (78.4% compared with 95.3%, respectively, P < 0.0004, Student's paired t test). The data presented in Figure 4b show that while slightly more than 60% ofoocytes progressed beyond metaphase I when cultured under controlled conditions, a very dramatic reduction in this proportion was observed in those groups exposed to caffeine concentrations greater than 2.4 mM. However, a marginal decrease in the proportion of post-metaphase I
314
Reproductive Toxicology
oocytes was revealed in those groups exposed to caffeine ranging between 0.00017 mM and 0.0017 mM, which was followed by an upward trend to 1.7 mM. Further analysis of the oocytes that had progressed beyond metaphase I revealed that they fell into three major groups: 1) those oocytes exhibiting a normal telophase I/metaphase II configuration; 2) those oocytes at diploid metaphase II; and 3) those oocytes that were aneuploid, but that did not exhibit a typical diploid metaphase II configuration (Figure 5). The proportion ofoocytes that contained normal telophase I-metaphase II configurations fluctuated between 0 m M and 0.0017 mM but underwent a progressive, albeit gradual, increase between 0.0017 and 1.7 mM that was followed by a dramatic rise between 1.7 mM and 5.1 mM. This trend was opposite to that observed for the proportion of post-metaphase I oocytes that were at diploid metaphase II; however, while the trend for aneuploid oocytes followed that for diploid metaphase II oocytes up to 0.0017 mM, and paralleled that for the proportion of normal telophase I-metaphase II oocytes between 0.0017 mM and 1.7 mM, it was diametrically opposed to that for normal mature oocytes between 1.7 mM and 10.2 mM. Of all aneuploid oocytes observed (n = 27), 81.48% were hyperhaploid, the incidence of hyperhaploidy being unrelated to the dose of caffeine tested. Since the proportion of meiotically arrested oocytes cultured for 4 h (Figure 3a) followed a trend similar to that for oocytes exhibiting normal telo-
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Volume 6. Number 4, 1992
phase I-metaphase II configurations following 20 h of culture (Figure 5), analyses were undertaken to determine the relevance of an initial period of meiotic arrest in cultured oocytes to completion of normal meiotic maturation. Given the relative lack of effect of caffeine in the maintenance of meiotic arrest at doses up to 0.017 mM, the data were analyzed in two groups: the first including oocytes cultured in the presence of 0 to 0.017 mM caffeine, and the second including oocytes cultured in caffeine at 0.17 to 10.2 mM. As shown in Figure 6, a positive correlation between the % GV and the % normal telophase I-metaphase II was observed for both groups, this relationship being statistically significant for oocytes exposed to the higher caffeine doses (0.17 to 10.2 mM; [r] = 0.987, P < 0.02, where [r] = the correlation coefficient). In contrast, negative correlations were observed between the % GV and the proportion of either diploid metaphase II or aneuploid oocytes, these correlations also being statistically significant only for the higher dose-range of caffeine tested ([ r] = - 0.988, P < 0.002, and [r] = -0.975, P < 0.05, respectively). Since less than 25% ofoocytes had resumed meiosis following 20 h of culture in the presence of 5.1 or 10.2 mM of caffeine (Figure 4), the possibility existed that these high doses of caffeine had an irreversible, and adverse, effect on oocyte meiotic competency. A series of experiments was conducted, therefore, in which the reversibility of caffeine-induced maintenance of meiotic arrest was investigated. Cumulusenclosed oocytes were cultured in the presence of 5.1 mM caffeine for 4 h, after which they were either airdried for analysis or transferred to fresh medium for an additional 4 h culture in the absence (control) or presence of 5.1 mM caffeine. When exposed continually to 5.1 mM caffeine, less than 20% of oocytes resumed meiosis, whether they were cultured for 4 or for 8 h (Table 1). In contrast, those oocytes transferred to control medium exhibited a high incidence of meiotic resumption with only 5% maintained at the GV stage.
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[Coffeine] mM Fig. 5. The effect o f caffeine on perturbations o f the first meiotic division in hamster oocytes. Following 20 h of culture in media containing various concentrations o f caffeine, oocytes were scored cytogenetically for the proportion that exhibited n o r m a l telophase I/metaphase II configurations (open diamonds), diploid metaphase II c h r o m o s o m e s (closed diamonds), or that were aneuploid (open triangles). Each value represents the m e a n + SEM o f 8 to 12 replicates.
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