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Regular Article |
a Department of Genetic Resources II, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan
b Department of Applied Genetics, Graduate School of Agriculture and Life Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
c Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-0810, Japan
d The Research Center for Protozoan Molecular Immunology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
ABSTRACT
Deterioration in the quality of mammalian oocytes during the metaphase-II arrest period is well known as "oocyte aging." Oocytes in which aging has occurred are called aged oocytes, and these oocytes show enhanced activation and higher fragmentation rates after parthenogenetic activation. Previously we showed that porcine aged oocytes had low maturation/M-phase promoting factor (MPF) activity, and we suggested that this low MPF activity contributed at least in part to the aging phenomena. In the present study, we examined the relationship between MPF activity and these aging phenomena by artificially regulating MPF activity in porcine metaphase-II-arrested oocytes. Since we have shown recently that aged porcine oocytes contain abundant phosphorylated inactive MPF, so-called pre-MPF, we used vanadate and caffeine, which affect the phosphorylation status of MPF, to regulate MPF activity. Incubation of 48-h-matured oocytes with vanadate for 1 h increased the phosphorylation of MPF and decreased MPF activity. The parthenogenetic activation and fragmentation rates were significantly increased compared with those of control oocytes. Conversely, treatment of 72-h-cultured aged oocytes with caffeine (last 10 h of culture) decreased the level of pre-MPF and elevated MPF activity. These oocytes revealed significantly lower parthenogenetic activation rates and a lower percentage of fragmentation than did untreated aged oocytes. These results indicate that not only the increased ability for parthenogenetic activation but also the increased fragmentation rate observed in porcine aged oocytes may be attributable in part to the gradual decrease in MPF activity during prolonged culture. Control of MPF phosphorylation with these agents may allow for some degree of manipulation of oocyte aging.
aging, meiosis
INTRODUCTION
Mammalian follicular oocytes freed from follicles can be matured in vitro, and their development is then arrested at metaphase-II until such time as the oocytes are penetrated by spermatozoa. Although maturation rates of porcine oocyte differ depending on the culture conditions (64%–94% [1]), nuclear status and morphologies of the matured oocytes do not change during this meiotic arrest. However, cytoplasmic changes affecting oocyte quality (for example, decreased ability to be fertilized and to develop) occur when the arrest period is prolonged [2, 3]. An increased tendency for spontaneous oocyte activation [4, 5] and subsequent fragmentation, an abnormal cleavage after activation characterized by unequal blastomeres [3, 5], has also been observed in porcine oocytes that were cultured for a long period. These quality changes occurring in metaphase-II-arrested oocytes during prolonged culture are called "aging," and those oocytes in which aging has occurred but that have not degenerated are called "aged oocytes." Aged and non-aged metaphase-II oocytes are identical in their morphology and differ only in quality; the molecular changes occurring in the cytoplasm and the mechanisms inducing these phenomena are not well understood.
Maturation/M-phase promoting factor (MPF) induces M-phase in eukaryotic cells, including oocytes [6]. We have already shown [5] that porcine aged oocytes, showing enhanced activation ability and higher rates of fragmentation, have decreased MPF activities. At present, this is the only known cytoplasmic difference between aged and non-aged oocytes. High MPF activity can be detected in metaphase-I and -II mammalian oocytes [7–10], and its inactivation in matured oocytes is induced by fertilization [7, 10–12] or parthenogenetic activation [5, 11, 13]. This abrupt inactivation of MPF has been considered to be the trigger for escape from metaphase-II arrest [14]. Therefore, the gradual decrease of MPF activity during aging is compatible with the gradual increase of cytoplasmic ability for oocyte activation in aged oocytes. Although the relationship between MPF activity and other aging phenomena, such as high fragmentation frequency, is unclear at present, low MPF activity might be one of the causes of the changes observed in aged oocytes.
Regulation of MPF activity depends upon the association of its catalytic subunit, p34cdc2 kinase, with its regulatory subunit, cyclin B, and the subsequent phosphorylation state at key tyrosine-15 (Y15) and threonine-14 (T14) residues [15]. In general, p34cdc2 is phosphorylated at T14 and Y15 by the Myt1 and Wee1 kinases after association with cyclin B, and this inactive form, called "pre-MPF," accumulates during G2-phase. Therefore, activation of MPF at the G2 to M transition depends on dephosphorylation at T14 and Y15 by cdc25 phosphatase [14]. Inactivation of p34cdc2 protein kinase is usually caused by proteolysis of cyclin B [15]. In mouse and porcine fertilized oocytes, degradation of cyclin B was clearly shown to be related to inactivation of p34cdc2 kinase [16, 17]. In contrast to these observations, we recently reported [17] that gradual phosphorylation of T14 and Y15 of p34cdc2 (in other words, accumulation of pre-MPF) occurred in aged oocytes. We suggested that in addition to the gradual decrease of cyclin B content in aged oocytes, this phosphorylation also contributed to the decrease in MPF activity. These findings prompted us to anticipate that artificial dephosphorylation of pre-MPF in aged oocytes might increase MPF activity and partially suppress the increased parthenogenetic activation ability of these cells.
In the present study, we tried to confirm our previous hypothesis that the increased ability with regard to parthenogenetic activation observed in porcine aged oocytes could be attributed in part to the gradual decrease of MPF activity of the oocytes during prolonged culture periods. For this purpose, we selected matured oocytes after culture by checking for the existence of a first polar body to avoid the contamination of immature oocytes, and we then used the matured oocytes to conduct the following experiments. Initially we examined whether vanadate, a potent inhibitor of tyrosine phosphatases, including cdc25 [18, 19] and caffeine (which has been reported to inhibit Myt1/Wee1 activity [20]), could change MPF activity in porcine oocytes arrested in the metaphase-II stage. Next, we studied whether changes in MPF activity could affect oocyte activation ability and fragmentation frequency, another well-known aging phenomenon, after parthenogenetic stimulation.
MATERIALS AND METHODS
We first checked the aging phenomena of the porcine oocytes cultured in vitro by evaluating nuclear changing and ability with regard to parthenogenesis. Next, we incubated oocytes with vanadate or caffeine to modify MPF activity. The efficiency of the oocytes was then checked using both MPF activity and phosphorylation status of p34cdc2 as measures; we also used parthenogenetic activation of the oocytes as an additional measure of efficiency.
Collection and Culture of Porcine Follicular Oocytes
Ovaries were obtained from prepubertal crossbred gilts (Landrace, Large White, and Duroc breeds) at a local slaughterhouse. Cumulus-oocyte complexes (COCs) were aspirated from nonatretic follicles that were 3–5 mm in diameter, and about 30 COCs were cultured in 500 µl of modified Waymouth MB752/1 medium (m-Waymouth) (Gibco BRL, Life Technologies, Inc., Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (Gibco BRL), 10% (v/v) porcine follicular fluid, 2.5 µg/ml FSH (Antrin; Denka Pharmaceutical Co. Ltd., Kanagawa, Japan), 100 IU/ml penicillin G potassium (Sigma Chemical Co., St. Louis, MO), and 0.1 mg/ml streptomycin sulfate (Sigma) at 39°C under air with 5% CO2, as previously reported [5, 17]. After culture for 24 to 72 h, some of the oocytes were examined for nuclear status using phase-contrast microscopy after fixation with acetic alcohol (1:3) and staining with 1% aceto-orcein solution.
Treatment of Metaphase-II-Arrested Oocytes with Vanadate or Caffeine
After maturation culture for 48, 60, and 72 h, the oocytes were denuded from cumulus cells by treatment with 150 IU/ml hyaluronidase and gentle pipetting. Metaphase-II-arrested oocytes were selected based on the presence of the first polar body (but no second polar body) under high magnification using a stereomicroscope (SZH-10; Olympus, Tokyo, Japan); oocytes were designated as 48-h, 60-h, and 72-h oocytes, respectively. Some of the 48-h oocytes were treated for the last 1 h with 500 µM sodium vanadate (Wako Pure Chemical, Tokyo, Japan) in m-Waymouth, and some of the 72-h oocytes were treated for the last 10 h with 5 mM caffeine (Sigma) in m-Waymouth.
Parthenogenetic Activation of Oocytes
Two kinds of stimulation were used in the present study. For weak stimulation, denuded oocytes were transferred to a hybridizing chamber (FTC-22W; Shimazu Corporation, Tokyo, Japan) containing 50 µl of activation solution (consisting of 0.3 M D-mannitol, 0.1 mM CaCl2, 0.1 mM MgCl2, and 0.2 mg/ml BSA). The oocytes were stimulated with a 20-µsec electric pulse at 1.0 kV DC/cm. For strong stimulation, oocytes were transferred to m-Waymouth containing 10 µM calcium ionophore A23187 (Sigma) and were incubated for 5 min. Both stimulated and nonstimulated oocytes were transferred to BMOC-II solution [21] and were subsequently cultured for 10 h and fixed. As controls, some oocytes were immediately fixed without parthenogenetic activation. Oocytes with a female pronucleus and fragmented oocytes were defined as activated [5]. At least three replicate trials were performed for parthenogenetic activation studies.
Assay of MPF Activity
For the assay of MPF activity, histone H1 phosphorylation activity (H1k activity) of the oocytes was assayed after immunoprecipitation using antiserum against the C-terminus of p34cdc2 [22]. Seventy-five oocytes suspended in 0.5 µl of 5x RIPA buffer were placed on a glass slide and ruptured using a coverslip. The lysate was recovered with fine-bore glass pipettes and incubated with 1 µl of antiserum against the C-terminus of mouse p34cdc2 [7] for 1 h at 4°C. Next, 5 µl of a 50% (v/v) slurry of protein A-agarose beads (Sigma) was added and incubated using a rotating wheel for 15 min at 4°C. The beads were washed three times in 0.5 ml of 1x RIPA buffer, added to 2.5 µl of assay solution (buffer A) [8] with 2.5 µM cAMP-dependent protein kinase inhibitor (TTYADFIASGRTGRRNAIHD; Sigma), and stored at -70°C until use. After thawing, 5 µl of 50 µM histone H1 (type III-S, Sigma) and 5 µl of 20 µM [(
-32P]ATP (3–10 cpm/fmol; Amersham, Arlington Heights, IL) were added, and the reaction was performed by incubating the mixture using a rotating wheel for 1 h at 36°C. The reaction was terminated by the addition of 5x SDS sample buffer (5 µl) followed by boiling for 5 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out [23] using 10% acrylamide gels, and phosphorylated histone bands were visualized after autoradiography. Three replicate trials were performed in this experiment.
Detection of Phosphorylation Status of p34cdc2
To determine the phosphorylated forms of p34cdc2, Western blotting analysis was conducted [22]. Fifty oocytes were boiled in 10 µl of SDS sample buffer for 5 min and then stored at -70°C until use. After thawing, SDS-PAGE was performed as described above. Proteins were transferred onto polyvinylidene-fluoride membrane (Immobilon-P, Millipore, Tokyo, Japan) using a Trans-Blot SD apparatus (Bio-Rad Laboratories, Hercules, CA) operated according to the manufacturer's instructions. The membrane was blocked by incubation for 1 h in Tris-buffered saline containing 0.1% Tween-20 and 5% dried milk before being incubated overnight with the anti-PSTAIRE monoclonal antibody [24]. Bound antibody was detected using a blotting detection kit (Amersham Pharmacia Biotech, Uppsala, Sweden), according to the manufacturer's instructions. The Western blotting was repeated at least three times.
The intensities of autoradiographs of H1k activity and the bands in Western blotting of p34cdc2 were analyzed by NIH Image (version 1.58; National Institutes of Health, Bethesda, MD).
Statistical Analysis
All data were subjected to analysis of variance using the GLM procedures of Statistical Analysis System (SAS Institute, Inc., Cary, NC). Least-squares means of the intensity of the bands in the Western blotting were analyzed using the least-squares means test after transformation using arcsine of percentage [25]. H1k activity ratios and parthenogenetic responses in percentages were transformed into arcsine, and they were analyzed by the Duncan multiple range test. The level of significance was set at P < 0.05.
RESULTS
Meiotic Maturation Stages of the Oocytes and Evaluation of Their Aging
In order to confirm that porcine oocytes cultured for a long period of time in our present maturation system are aged oocytes, the meiotic maturation stages of oocytes cultured for 24–72 h and the degree of oocyte aging were examined. Matured oocytes at metaphase-II were first observed after 30 h of culture, and the maturation rate increased to 55% at 36 h; thereafter, the rate was almost constant, at about 65%, until 72 h of culture (Fig. 1). The incidences of spontaneous oocyte activation and oocyte degeneration were less than 3% and 9%, respectively, throughout the culture period. An example of a 72-h oocyte arrested at metaphase-II is shown in Figure 2, C and D. Its morphology was not distinguishable from that of 48-h oocytes (Fig. 2, A and B).
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The degree of oocyte aging was evaluated by both oocyte activation and fragmentation rates, which were examined after an additional 10 h of culture with or without weak stimulation [5]. The spontaneous activation rates of 36- to 60-h oocytes were almost zero after an additional culture (total: 46–70 h culture), but that of 72-h oocytes (total: 82 h culture) was elevated significantly. This trend was more clearly evident after weak stimulation for parthenogenetic activation, in which case the incidence of oocyte activation significantly increased as the duration of the culture period was prolonged (Fig. 3A). The morphologies of the activated 72-h oocytes are shown in Figure 2, E–H. The oocytes that formed a female pronucleus were morphologically normal (Fig. 2, E and F), but some oocytes were fragmented (as shown in Fig. 2, G and H), characterized by unequal cleavage with small blastomeres. Most of the blastomeres did not have a nucleus. Fragmentation of oocytes was not observed in 36-h or 48-h oocytes with or without stimulation, and the rate was increased in both stimulated and unstimulated oocytes cultured for more than 60 h (Fig. 3B). These results indicate that oocyte aging gradually progressed in our culture system, and we consider oocytes cultured for more than 60 h to be aged oocytes.
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Effects of Vanadate and Caffeine on the Phosphorylation Status in p34cdc2 of Porcine Oocytes
Changes in phosphorylation status of p34cdc2 were detected by three migrating bands (named U, M, and L, from the slowest to the fastest migrating bands, respectively) upon immunoblotting with anti-PSTAIRE antibody. It has been reported that p34cdc2 has three phosphorylation sites: T14, Y15, and threonine-161 (T161). In pre-MPF, p34cdc2 is phosphorylated at T14 and/or Y15, the inhibitory phosphorylation sites, and such p34cdc2 molecules migrate in the U and M bands, whereas p34cdc2 dephosphorylated at both of these sites migrates in the L band [26, 27]. Therefore, the intensity of the U and M bands was considered to reflect the level of pre-MPF. As the content of p34cdc2 was almost constant throughout the culture period, we used the intensity ratio of the U+M bands to the L band to assess the relative level of pre-MPF.
A typical example of an immunoblot of p34cdc2 and the relative pre-MPF level in porcine oocytes, as calculated by analysis with NIH Image, are shown (respectively) in Figure 4, A and B. We reported previously that the level of cyclin B was extremely low in porcine germinal vesicle oocytes and increased after germinal vesicle breakdown, when cyclin B synthesis begins [22]. In strong agreement with the cyclin B level in maturing oocytes, a significant amount of pre-MPF was detected in metaphase-II-arrested porcine oocytes cultured for 48 h, whereas the amount was quite low in germinal vesicle oocytes (0 h culture). A gradual increase in pre-MPF content was observed during culture from 48 to 72 h, and the amount in metaphase-II-arrested oocytes cultured for 72 h was significantly higher than that found in 48-h oocytes. When 48-h oocytes were treated with vanadate for the last 1 h of culture, the amount of pre-MPF increased significantly and was almost the same as that observed in 72-h oocytes. In contrast, when 72-h oocytes were treated with caffeine for the last 10 h of culture, the amount of pre-MPF decreased significantly and was almost the same as that observed in 48-h oocytes. These results indicate that inhibition of cdc25 or Myt1/Wee1 affects the equilibrium of MPF and pre-MPF in porcine metaphase-II-arrested oocytes and that the relative amount of pre-MPF can be artificially controlled, at least in part.
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Effects of Vanadate and Caffeine on H1 Phosphorylation Activities of Porcine Oocytes
We next examined whether artificial modification of p34cdc2 phosphorylation status by vanadate and caffeine also changed H1k activity in metaphase-II-arrested porcine oocytes. Example of an autoradiograph of the H1k activity assay and the activity level calculated after analysis with NIH Image are shown (respectively) in Figure 5, A and B. In germinal vesicle oocytes (0 h of culture), H1k activity was low and measured about fourfold greater after maturation for 48 h. The high H1k activity in 48-h oocytes decreased significantly as the maturation period was prolonged up to 72 h, as reported previously [5]. When the dephosphorylation of pre-MPF in 48-h oocytes was inhibited and the amount of pre-MPF was increased to the level in 72-h oocytes via vanadate treatment, H1k activity was significantly less than that observed in nontreated oocytes, although the H1k activity in vanadate-treated 48-h oocytes was significantly greater than that of 72-h oocytes. On the other hand, a decrease in the level of pre-MPF in 72-h oocytes to the 48-h oocyte level following caffeine treatment resulted in a significant elevation of H1k activity, although the H1k activity was significantly less than that observed in 48-h oocytes. These results indicate that artificial modification of the equilibrium of MPF and pre-MPF in porcine metaphase-II-arrested oocytes affects their H1k activities significantly and that vanadate and caffeine may be used to regulate MPF activity in porcine oocytes.
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Effects of Vanadate and Caffeine on Aging Phenomena in Metaphase-II-Arrested Porcine Oocytes
The effects of partial regulation of MPF activity on aging phenomena in metaphase-II-arrested porcine oocytes were examined next. The parthenogenetic activation rates (Fig. 6A) of 48-h and 72-h oocytes immediately after culture were low (0% and 11%, respectively), and no significant effects of vanadate and caffeine treatment on the activation rate were observed at this time point (immediately after treatment). When these oocytes were cultured for an additional 10 h, however, the activation rate of 72-h oocytes was significantly higher than that of 48-h oocytes (65% vs. 7%). In 48-h oocytes, suppression of MPF activity by vanadate significantly increased the rate (42%). In contrast, in 72-h oocytes, elevation of MPF activity by caffeine significantly decreased the rate (38%).
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The fragmentation rates (Fig. 6B) were examined after strong stimulation of oocytes by ionophore treatment and culture for an additional 10 h. Activation rates exceeded 82% in all groups (data not shown). Low and high fragmentation rates were observed in 48-h and 72-h oocytes (3% and 57% of activated oocytes, respectively). The low fragmentation rate of 48-h oocytes was significantly increased to 27% when we decreased their MPF activity via vanadate treatment. On the other hand, elevation of MPF activity in 72-h oocytes via caffeine treatment resulted in a significant decrease in the fragmentation rate (to 18%). These results indicate that the decrease of MPF activity in metaphase-II-arrested porcine oocytes contributes not only to the enhanced ability for oocyte activation but also to the increased frequency of fragmentation.
DISCUSSION
In the present study, we confirmed the hypothesis that we had proposed previously [5] that the increased ability for parthenogenetic activation observed in porcine aged oocytes could be attributed in part to the gradual decrease of MPF activity of the oocytes during prolonged culture. Furthermore, the present study also suggests that this decrease in MPF activity contributes to the increased fragmentation rate of porcine aged oocytes. Although these events in aged oocytes have been known for almost 20 years [3], this is the first report that relates them to cytoplasmic changes and provides a molecular basis for the phenomenon of oocyte aging.
Mechanism for the Decrease of MPF Activity in Porcine Aged Oocytes
Recently we have shown that aged porcine oocytes contain abundant MPF that is inactivated by phosphorylation—so-called pre-MPF—and we suggested that the decrease in MPF activity in porcine aged oocytes could be attributed in part to the accumulation of pre-MPF via the imbalance of kinase and phosphatase activities [17]. This suggestion was confirmed by the present results, which show significant elevation of MPF activity in aged oocytes after artificial dephosphorylation at T14 and Y15 of p34cdc2 as a result of caffeine treatment. Caffeine has been reported to cause an induction of the T14 and Y15 dephosphorylated form of p34cdc2, thereby resulting in an elevation of MPF activity, in mammalian cultured cells [28, 29] and Xenopus oocytes [20]. Although the target of caffeine has not been defined, the inhibitory action has been suggested to be aimed at tyrosine kinases such as Myt1/Wee1, which accelerate p34cdc2 kinase phosphorylation at T14 and Y15, thereby resulting in the accumulation of pre-MPF [20]. Therefore, the present effect of caffeine on the elevation of MPF activity might also be inhibition of Myt1/Wee1 kinase rather than stimulation of cdc25 phosphatase, which is an activator of pre-MPF [30].
Vanadate is an established inhibitor of protein tyrosine phosphatases, including cdc25 [18, 19]. The inhibitory effects of vanadate on the transition of pre-MPF to active MPF have been shown in meiotic resumption of mouse [31], rat [32], and pig [33] oocytes and also in the meiotic arrest (at metaphase-II) of pig oocytes (present study). These results also support the view that preservation of the balance of Myt1/Wee1 kinase and cdc25 phosphatase activities is important for the maintenance of MPF activity in metaphase-II-arrested porcine oocytes. The equilibrium of MPF and pre-MPF in metaphase-II-arrested porcine oocytes appears to move gradually in the direction of pre-MPF during a prolonged culture period. Vanadate induces this change, and the phosphorylation state of p34cdc2 in non-aged oocytes shifts toward that in aged oocytes. On the other hand, caffeine alters the equilibrium in the direction of active MPF and subsequently, the phosphorylation state of p34cdc2 in aged oocytes shifts to that observed in non-aged oocytes.
The MPF activity of caffeine-treated 72-h oocytes was significantly lower than that of 48-h oocytes, and conversely, the MPF activity of vanadate-treated 48-h oocytes was significantly higher than that of 72-h oocytes. As the pre-MPF levels in both types of oocytes were almost the same, these differences in MPF activity should be attributed to differences in cyclin B content between 48-h and 72-h oocytes. These results show that not only the imbalance of Myt1/Wee1 and cdc25 activities but also a gradual decrease of cyclin B content contributes to the decreased MPF activity in porcine aged oocytes. The possible mechanisms for decreased MPF activity in porcine aged oocytes and the effects of vanadate and caffeine are schematically illustrated in Figure 7.
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MPF Activity Relates to Aging Phenomena in Porcine Oocytes
In order to test the hypothesis that the low MPF activity in aged porcine oocytes contributes to a high parthenogenetic activation rate [5], MPF activity in metaphase-II-arrested oocytes was artificially regulated with vanadate and caffeine treatment, and the resulting parthenogenetic activation rates were examined. The results show that elevation and depression of MPF activity correlate with decrease and enhancement, respectively, of spontaneous activation rate. These results agree well with our hypothesis and define the relationship between MPF activity and parthenogenetic activation ability. An interesting finding was the relationship between MPF activity in metaphase-II-arrested oocytes and the rate of fragmentation after activation. These results strongly indicate that high MPF activity before activation is necessary for normal female pronuclear development and that the high frequency of fragmentation in aged oocytes is attributable to their low MPF activity. Sato et al. [34] have reported that the addition of dibutyryl cAMP to the culture medium significantly increased the incidence of fragmented oocytes after 72 h of culture. The inhibitory effect of cAMP on activation of MPF in meiotic arrested oocytes has been firmly established [35–37]. Therefore, the effects of dibutyryl cAMP on the induction of fragmented oocytes might be mediated by inhibition of pre-MPF dephosphorylation and depression of MPF activity in the oocytes.
The molecular mechanisms that induce fragmentation by low MPF activity are unknown at present. It has been reported that MPF phosphorylates the microtubule-associated protein p220, which binds tubulin and affects microtubule formation [38, 39], as well as the intermediate filament protein vimentin [40]. Therefore, one possible explanation is that MPF changes the organization of cytoskeletal filaments and affects their function and the subsequent cytokinetics of the oocyte. However, these observations have been reported in in vitro studies, and there is very little evidence to explain the biological effects of MPF in vivo. Furthermore, the mechanisms of fragmentation in mammalian oocytes (for example, its relationship to the function of cytoskeletal filaments) have not been studied. Further experiments are needed to elucidate the connection between MPF activity and oocyte fragmentation.
The frequencies of parthenogenetically activated oocytes and fragmented oocytes were almost identical in vanadate-treated 48-h oocytes and caffeine-treated 72-h oocytes, although the MPF activity in the former was much higher than in the latter. This result clearly indicates that other factors (apart from MPF activity) are involved in these aging phenomena. One possible candidate is the amount of pre-MPF, which increases during the culture period and which might promote aging of oocytes. Our current hypothesis is that the ratio of active MPF to pre-MPF shows a higher correlation with aging phenomena in porcine oocytes.
Practical Advantage of Caffeine Treatment in Oocyte Manipulation
In vitro matured oocytes are widely used in advanced reproductive technologies such as in vitro fertilization, sperm injection, and cloning by nuclear transfer. Control of oocyte aging might have many advantages for these procedures, since limitations on manipulation time could be removed and oocyte quality could be refined. As a result, establishment of methods for aging control might assist progress in these technologies. The present report is the first to show that caffeine treatment causes a significant decrease of aging phenomena in oocytes cultured for a long period; furthermore, this treatment is very simple to apply. Although extensive consideration of safety aspects will be required prior to practical application, this methodology might be useful in reproductive technology.
ACKNOWLEDGMENTS
The authors thank Dr. D.A. Vaughan for the improvement of the textual English and Ms. T. Aoki and Ms. E. Yamauchi for technical assistance.
FOOTNOTES
1 Supported by a grant-in-aid for scientific research (10660267 and 11556051 to K.N.; 08406018, 09876073, and 10356010 to H.T.) from the Ministry of Education, Science, Sports and Culture of Japan. 
2 Correspondence: Kazuhiro Kikuchi, Laboratory of Animal Conservation, Department of Genetic Resources II, National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. FAX: 81 298 38 7408; kiku{at}abr.affrc.go.jp
Accepted: April 11, 2000.
Received: January 24, 2000.
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