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Gamete Biology |
a Laboratory of Animal Reproduction, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8528, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cumulus cells, gamete biology, meiosis, ovum, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many of the factors secreted by gonadotropin-stimulated cumulus cells of cumulus-oocyte complexes (COCs) can affect the meiotic maturation of oocytes [9–11]. Meiosis-activating sterol, FF-MAS, which was first reported to be purified from human follicular fluid (4,4-dimethyl-5
-cholesta-8,14,24-trien-3ß-ol), has been synthesized by COCs in response to FSH stimulation [10, 12]. This type of sterol is an intermediate in the cholesterol biosynthetic pathway produced by the demethylation of lanosterol by the cytochrome P450 enzyme, 14-demethylase [13]. Byskov et al. [12] showed that FF-MAS can induce in vitro meiotic maturation of mouse denuded oocytes in a dose-dependent manner, and Faerge et al. [14] have reported the distribution of [3H]FF-MAS binding sites by using transmission electron microscopic autoradiography in the oocytes of marmosets, cows, and mice. The results by Faerge et al. showed that specific binding of FF-MAS is predominant at the oolemma of denuded oocytes, suggesting the existence of a plasma membrane-associated molecule with affinity for FF-MAS. However, there have been no reports describing the isolation and cloning of the FF-MAS receptor.
Downs et al. [15] also reported the simulative activity of FF-MAS on denuded oocytes; however, they showed that meiosis was not induced by FF-MAS in cumulus-enclosed oocytes. When rat follicles were cultured with ketoconazole, an inhibitor of FF-MAS synthesis, it had no effect on meiotic resumption, whereas LH-induced progesterone synthesis was suppressed by this drug [16]. Moreover, the addition of AY9944, an inhibitor of the cholesterol synthetic pathway of FF-MAS, caused a decrease in LH-induced progesterone production and suppression of meiotic resumption of oocytes in rat follicles [17]. These results suggest that synthesis of the sterol by FSH-stimulated cumulus cells did not accumulate, but instead was metabolized into cholesterol, although FF-MAS had a potentially positive function in the meiotic resumption of oocytes. This idea has been supported in a report by Baranao and Hammond [18] that FSH augmented cholesterol synthesis in granulosa cells.
Cytochrome P450scc, which cleaves the side chain of cholesterol, is activated in FSH-stimulated cumulus cells that surround oocytes [8, 19, 20]. Lieberman et al. [21] showed that meiosis-inducing action of LH was not suppressed by P450scc inhibitor, aminoglutethimide (AGT) in rat follicles. However, Osborn et al. [22] reported that when ovine follicles were cultured with FSH, LH, and AGT, the result was an almost complete inhibition of progesterone production and meiotic progression to the metaphase II stage in oocytes. In our previous study [8], during in vitro maturation of porcine COCs, the addition of AGT significantly suppressed progesterone production by COCs and GVBD in cumulus-enclosed oocytes. In gilts after the LH surge, meiotic resumption occurs in oocytes, which is followed by an increase in progesterone concentrations in follicular fluid [23, 24]. These cumulative findings have revealed that the production of steroid hormones from cholesterol, especially progesterone, by cumulus cells being stimulated by FSH, LH, or both is required for meiotic resumption of porcine oocytes. In porcine and bovine oocytes, addition of progesterone into a maturation medium stimulates meiotic resumption, regardless of the presence or absence of gonadal hormones [25, 26]. However, other reports have shown no statistically significant stimulation of meiotic resumption by progesterone in porcine oocytes [27]. Moreover, progesterone has been reported to suppress spontaneous meiotic maturation in mouse oocytes [28]. Thus, it is unclear whether or not progesterone is required for in vitro meiotic resumption in mammalian oocytes.
Hormone levels secreted by COCs in culture medium, such as those of progesterone, may rise with an increase in the number of COCs in each culture well. Therefore, we hypothesized that cultivation of one COC in each well would be insufficient to produce a high amount of the hormones in the medium of each well; the low amount results in delaying meiotic resumption. In this study we investigated the effects of the number of COCs in the maturation medium on GVBD progression, and the effects on the rate of GVBD of additional progesterone in the medium when one COC was in each well for 20 h. In addition, we demonstrated the role played by sterol biosynthesis in progesterone production by cumulus cells and in oocyte GVBD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Porcine ovaries were collected from 5- to 7-mo-old prepubertal gilts at a local slaughterhouse and transported within 1.5 h to the laboratory in 0.85% (w/v) NaCl containing 0.1 mg/ml kanamycin (Meiji Seika, Tokyo, Japan) at about 30°C. The surfaces of intact, healthy antral follicles measuring 3–8 mm in diameter were cut with a razor blade and oocytes were collected with a surgical blade to scrape the inner surface of follicle walls. The collected oocytes were placed in prewarmed PBS pH 7.4 supplemented with 0.1% (w/v) polyvinyl-pyrrolidone (Sigma Chemical Co., St. Louis, MO). We used a stereomicroscope to select oocytes with evenly granulated cytoplasm and at least four layers of unexpanded cumulus oophorus cells that were not attached with mural granulosa cells. They were washed three times with maturation medium. Twenty COCs were cultured in each well of a 48-well multidish (Nunc, Roskilde, Denmark) containing 300 µl of maturation medium at 39°C in a humidified atmosphere of 5% CO2 in air. The basic maturation medium was modified North Carolina State University 37 [29] containing 10% (v/v) fetal calf serum (Gibco BRL, Grand Island, NY), 0.6 µg/ml of porcine FSH (Sigma), 1.3 µg/ml of equine LH (Sigma), and 7 mM Taurine (Sigma).
Assessment of Nuclear Maturation
After incubation, the oocytes were freed from cumulus cells, then mounted on slides, fixed with acetic acid/ethanol (1:3) for 48 h, and stained with aceto-lacmoid before examination under a phase-contrast microscope (400x) in order to evaluate their chromatin configurations.
Quantification of Progesterone in Medium by HPLC-UV Analysis
Quantification of progesterone by HPLC-UV was based on the procedures reported by our previous study [8]. Briefly, the medium in which COCs had been cultured was collected into plastic tubes and centrifuged at 10 000 x g for 20 min. Progesterone was extracted from the medium by 5-min mixing with 10 ml of dichloromethane (Nakalai, Kyoto, Japan). After centrifugation, the 10-ml dichloromethane fraction was collected into a disposal tube and the solvent from this fraction was removed by vacuum extraction for 120 min at 5°C. Samples were reconstituted in 100 µl of 50% (v/v) methanol solution. The samples were separated using a reverse-phase CAPCELL PAK column (2 x 100 mm) (Shiseido, Tokyo, Japan). The solvent delivery system contained a 50% (v/v) methanol solution. Detection of progesterone was performed at 240 nm using a UV detector and peak heights were measured using a computer integrator.
The standard curve of progesterone was linear from zero to 800 ng/ml. The intraassay coefficient of variation (CV) in medium with 100 ng/ml of progesterone was 4.15%. The recovery rate for 50 ng of progesterone added to 0.5 ml of the medium (100 ng/ml) was 93.8% ± 5.4%.
Statistical Analysis
Statistical analyses of all data from three or four replicates for comparison were carried out with one-way ANOVA followed by the Duncan multiple range test using Statview (Abacus Concepts, Inc., Berkeley, CA). All percentage data were subjected to arc-sine transformation before statistical analysis.
Experimental Design
In experiment 1 we investigated the relationship between adding COCs to each well and the time period in which GVBD was exhibited. After collected COCs were washed three times with maturation medium, 1, 5, 10, or 20 COCs were transferred to each well containing 300 µl of the medium. The COCs cultured for 20, 24, or 28 h in each well were denuded and assessed for their nuclear status. Cultured medium was used to assess progesterone levels as described above.
In experiment 2, to investigate the effects on GVBD of the addition of progesterone into the medium, one COC was transferred to each well containing the maturation medium supplemented with or without 20 ng/ml of progesterone. After the COCs had been cultured for 20 h in each well, they were denuded and their nuclear status was assessed. Because the progesterone level in the medium in which 20 COCs had been cultured for 20 h was about 20 ng/ml, the additional concentration of progesterone was 20 ng/ml in the maturation medium in this experiment.
In experiment 3 we examined the effects of suppressing meiosis-activating sterol production on progesterone production by FSH- and LH-stimulated cumulus cells and meiotic resumption of oocytes. COCs were cultured with 1 or 10 µM of a 14-demethylase inhibitor, ketoconazole (Sigma), for 20 or 28 h. COCs were denuded and their nuclear status was assessed. The culture medium was the same as that used to analyze progesterone as described above. To investigate the effects on the resumption of meiosis when additional progesterone was introduced into the ketoconazole-containing medium, 20 COCs were cultured with both 10 µM ketoconazole and 15 ng/ml of progesterone. Because the progesterone level in the medium in which 20 COCs had been cultured for 20 h without ketoconazole was about 21.8 ng/ml and that in the medium with 10 mM ketoconazole was 6.5 ng/ml, the additional concentration of progesterone was 15 ng/ml in maturation medium. Ketoconazole was dissolved in ethanol at 50 mM. Final concentrations (1 or 10 µM) were obtained by dilution with each maturation medium. As a control, inhibitor-free medium was prepared by adding 0.1% (v/v) ethanol to the basic maturation medium.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of the number of COCs in the maturation medium on GVBD progression One, 5, 10, or 20 COCs were transferred to each well containing 300 µl of maturation medium and then cultured for 20, 24, or 28 h. When one COC was cultured in each well for 20 h, the GVBD rate was 30.0% ± 1.9%. The GVBD rate was significantly greater with an increase in the number of COCs cultured in each well (Fig. 1). A significant positive correlation between the GVBD rate and the number of COCs cultured in each well for 20 h was observed (r = 0.8994; P < 0.05) (Fig. 2).
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After a 24-h cultivation period, the proportion of GVBD-completed oocytes in the group with one COC was significantly lower than that of COC oocytes cultured with 5, 10, or 20 COCs in each well (Fig. 1). No significant difference in GVBD was observed when 5, 10, or 20 COCs were cultured in each well for 24 h (Fig. 1).
When COCs were cultured for 28 h, more than 70% of the oocytes exhibited GVBD. A significantly lower rate of GVBD was detected in oocytes when COCs were cultured alone compared with the rate that occurred when 10 COCs were cultured in each well; however, no significant differences in GVBD rates were observed when 1, 5, or 20 COCs were cultured for 28 h (Fig. 1).
Effects of the number of COCs in maturation medium on progesterone levels in the medium After 1, 5, 10, or 20 COCs were cultured for 20 h, the progesterone concentration in the culture medium was analyzed by the HPLC-UV method. The level of progesterone in the medium in which one COC had been cultured for 20 h was 1.03 ± 0.211 ng/ml. Progesterone concentrations rose significantly with an increase in the number of COCs cultured (Table 1). When 20 COCs were cultured in each well, a maximum level of 21.75 ± 5.035 ng/ml of progesterone in the culture medium was detected.
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The amount of progesterone produced by one COC was in the narrow range of 0.290 ± 0.087 ng to 0.337 ± 0.055 ng. The different levels in progesterone production per single COC cultured with 1, 5, 10, or 20 COCs in each well were not significant (Table 1).
After COCs were cultured with 1, 5, 10, or 20 COCs in each well, the GVBD rate of oocytes cultured in each well and the level of progesterone in each well were analyzed. The correlation between the GVBD rate of oocytes cultured in each well and the progesterone concentration in each well was significant (r = 0.9384; P < 0.01) (Fig. 3).
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Experiment 2
The results of experiment 1 showed that increasing the number of COCs in each well induced a high level of progesterone in the medium and that it also accelerated GVBD. The purpose of this was to examine whether or not a high amount of progesterone in the medium would induce an acceleration of GVBD in these oocytes.
When one COC was cultured in each well containing medium without progesterone for 20 h, a low GVBD rate (25.6% ± 5.1%) was observed. The addition of 20 ng/ml of progesterone to the medium in which 20 COCs were cultured for 20 h caused a significant rise in GVBD rates in these oocytes (Fig. 4). This proportion of GVBD (73.6% ± 9.0%) was entirely comparable to that in COCs that were cultured in the absence of additional progesterone with 20 COCs (69.1% ± 2.1%).
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Experiment 3
After 20 COCs were cultured in the basic maturation medium for 20 h, the majority of oocytes exhibited GVBD. The addition of 1 or 10 µM ketoconazole to the medium significantly suppressed meiotic resumption in oocytes (Fig. 5). The GVBD rate was lower in oocytes that were cultured with 10 µM ketoconazole compared with that of oocytes cultured with 1 µM of this drug; however, this difference was not significant (Fig. 5). The low GVBD rate in oocytes when COCs were cultured with 10 µM ketoconazole rose significantly with an additional 8-h cultivation period (Fig. 5). The resulting rate was fully comparable to that of oocytes cultured for 20 h without the drug (control) (Fig. 5).
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The level of progesterone in the medium in which COCs had been cultured for 20 h was 21.75 ± 5.035 ng/ml (Fig. 6). The level was significantly inhibited by the addition of 10 µM ketoconazole (6.45 ± 0.701 ng/ml) (Fig. 6).
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To investigate the effects on meiotic resumption of additional progesterone in the ketoconazole-containing medium, 20 COCs were cultured with both 10 µM ketoconazole and 15 ng/ml of progesterone. The addition of progesterone resulted in a greater proportion of oocytes resuming with meiosis (Fig. 5); this result indicates that the inhibitory effect of ketoconazole on meiotic resumption was overridden by the addition of progesterone.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been reported that progesterone is secreted by COCs during in vitro maturation, and that the level of progesterone increases when stimulated with LH, FSH, or forskolin in pigs [7–9, 32], mice [33], and cattle [34]. In amphibian oocytes, progesterone was also synthesized in pituitary hormone-stimulated follicle cells [35, 36]; the secreted progesterone acts at the oocyte surface [37], which reduces the cAMP level in oocytes through the activation of phosphodiesterase, which then induces meiotic resumption [38]. In fish, it has been reported that progestin stimulates GVBD in oocytes [39]. On the other hand, in mouse oocytes, phosphodiesterase activity was suppressed by the addition of progesterone to the medium [40]. In porcine denuded oocytes, no statistically significant stimulation of meiotic resumption by progesterone has been observed [27], however, there is little information about the role of progesterone in meiotic resumption of cumulus cell-enclosed oocytes. In the present study, the level of progesterone in the culture medium rose with an increase in the number of porcine COCs in each well. Furthermore, a significantly lower rate of GVBD occurred in oocytes when one COC was cultured in each well for 20 h, but it improved when more progesterone was added to the medium; this rate was comparable to that observed in oocytes when 20 COCs were cultured in each well for 20 h. Thus, the results of the present study support our hypothesis that progesterone secreted by COCs plays a role in inducing GVBD in porcine cumulus-enclosed oocytes.
Recently, Shimada and Terada [8] have shown that the addition of LH and FSH induces progesterone receptor formation in cumulus cells concomitantly with greater progesterone production by COCs. They also showed that the addition of progesterone to medium without LH and FSH did not accelerate GVBD in porcine oocytes [8]. Judging from these results, Shimada and Terada [8] estimated that the binding of progesterone to its receptor in cumulus cells, which was secreted by LH- and FSH-stimulated cumulus cells, was associated with GVBD induction in porcine oocytes. Thus, this report suggests that different mechanisms of progesterone-induced meiotic resumption exist between amphibian and mammalian oocytes; in amphibian oocytes progesterone acts at the oocyte surface, and in mammalian oocytes (or at least in porcine oocytes), progesterone stimulates meiotic resumption through the action of the progesterone receptor in cumulus cells.
FF-MAS purified from human follicular fluid was shown to stimulate meiotic maturation of human and mouse oocytes [12]. Addition of an inhibitor of FF-MAS synthesis, ketoconazole, resulted in an inhibition of GVBD in mouse oocytes in a dose-dependent fashion [41]. However, Downs et al. [15] were unable to demonstrate any effects of ketoconazole on the suppression of meiotic resumption in mouse cumulus-enclosed oocytes when COCs were cultured with FSH and hypoxanthine. The present results showed that a 28-h cultivation of COCs resulted in no significant difference in GVBD rate of oocytes cultured in the presence or absence of ketoconazole, whereas the GVBD rate in oocytes cultured with ketoconazole for 20 h was significantly lower than it was without the drug. These findings imply that sterol biosynthesis from lanosterol in cumulus cells is involved in the acceleration of GVBD in mammalian oocytes.
Tsafriri et al. [16] reported that when rat antral follicles were cultured for 24 h with LH and 10 µM ketoconazole, the progesterone concentration in follicular fluid was significantly decreased, whereas 10 µM ketoconazole did not significantly affect oocyte meiotic resumption. In the present study, the addition of 10 µM ketoconazole to the medium significantly suppressed not only ongoing GVBD but also progesterone production in COCs after a 20-h cultivation period. FF-MAS is an intermediate in the cholesterol biosynthetic pathway [13], and progesterone is known to be produced from cholesterol in cumulus cells during in vitro maturation [8, 19, 20]; therefore, we hypothesized that FF-MAS is metabolized into cholesterol and then converted to progesterone in cumulus cells. In the present study, when COCs were cultured with ketoconazole and progesterone for 20 h, the rate of GVBD in oocytes was similar to that in oocytes cultured without ketoconazole. Judging from these results, we estimate that FF-MAS is synthesized in cumulus cells and does not accumulate, but is metabolized to cholesterol, which is then converted into progesterone; the secreted progesterone then accelerates ongoing GVBD in porcine oocytes. The metabolic pathway from FF-MAS to cholesterol, and its conversion to progesterone in cumulus cells during in vitro maturation of oocytes, as well as the physiological roles played by these factors in oocyte maturation are now under investigation.
In conclusion, increasing the number of COCs in each well caused an acceleration of ongoing GVBD in oocytes and induced an increase in the concentration of progesterone in the medium. A significant positive correlation between the level of progesterone and the proportion of oocytes undergoing GVBD was observed when COCs were cultured for 20 h. A significantly low rate of GVBD in oocytes when one COC was cultured in each well for 20 h was improved by transiently adding progesterone to the medium. Furthermore, the addition of ketoconazole to the medium suppressed the rate of GVBD as well as the progesterone production in COCs cultured for 20 h. However, the suppression of ongoing GVBD by ketoconazole was countered by additional progesterone. These results showed that the high level of progesterone, which was produced in cumulus cells from cholesterol, is responsible for the acceleration of ongoing GVBD in porcine oocytes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Masayuki Shimada, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8528, Japan. FAX: 81 824 24 7988; mashimad{at}hiroshima-u.ac.jp
Received: 4 September 2002.
First decision: 17 September 2002.
Accepted: 16 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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