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Gamete Biology |
Bernhard Zondek Hormone Research Laboratory, Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-demethylase (LDM). Follicular LDM was preferentially localized in oocytes by immunohistochemistry. Using [3H]acetate or R-[5-3H]mevalonate as precursors as well as high-performance liquid chromatographic and thin-layer chromatographic separation, we have measured the concentrations of de novo-synthesized lanosterol, FF-MAS, and cholesterol in rat graafian follicles, cumulus-oocyte complexes (COCs), and denuded oocytes (DOs) treated with LH, AY-9944 (an inhibitor of 
14-reductase, which was anticipated to increase FF-MAS levels by inhibiting its metabolism), or both after 8 h of culture. In follicles, both LH and AY-9944 increased the accumulation of FF-MAS as compared to controls. In COCs, AY-9944 caused a marked increase in FF-MAS, but we were unable to detect accumulation of FF-MAS in DOs. Neither the endogenous increases in FF-MAS accumulation nor the addition of FF-MAS to the culture medium could overcome the inhibition on resumption of meiosis by phosphodiesterase inhibitors. Compared to LH-induced resumption of meiosis in follicles, that induced by AY-9944 was much delayed. These results call into question any role of FF-MAS as an obligatory mediator of LH activity on germinal vesicle breakdown. The discrepancy between the positive staining for LDM in oocytes and our inability to detect de novo synthesized FF-MAS in DOs may relate to the sensitivity of the methodology employed and either the number of oocytes used or a deficiency in LDM synthetic activity in such oocytes. Further studies are required to confirm any of these alternatives.
cumulus cells, follicle, gamete biology, meiosis, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Culture of mouse isolated oocytes with hypoxanthine, an inhibitor of the spontaneous resumption of meiosis, suggested that a positive regulator produced by somatic cells is transported to the oocyte and induces its maturation [3– 6]. Using the same model system, Byskov et al. [7] identified this activity as a sterol. The putative regulatory sterol in human follicular fluid (follicular fluid meiosis-activated sterol [FF-MAS]) was identified as 4,4-dimethyl-5-cholest-8,14,24-triene-3ß-ol and that from bull testicular tissue (T-MAS) as 4,4-dimethyl-5-cholest-8,24-diene-3ß-ol. Both FF-MAS and T-MAS modestly increased the proportion of maturing mouse oocytes cultured with hypoxanthine as an inhibitor [7]. The FF-MAS is the product of demethylation of lanosterol by lanosterol 14-demethylase (LDM) and is converted to T-MAS by a sterol 14-reductase [8, 9]. Activity of LDM is stimulated in immature rat ovaries by eCG, resulting in a sixfold increase within 48 h [10].
The ability of MAS to cause resumption of meiosis in the presence of hypoxanthine was confirmed by several investigators [11–13]. Therefore, it has been suggested that FF-MAS is a signal molecule transducing the meiosis-inducing action of gonadotropins from the granulosa cells to the oocyte [14–16]. This idea was not supported, however, by experiments in which inhibitors of LDM failed to block either spontaneous or LH-induced oocyte maturation [17]. In addition, it was not established which cells in the ovary are responsible for the gonadotropin-induced MAS accumulation. Immunohistochemistry localized LDM preferentially in the oocytes [17]. However, AY-9944, an inhibitor of 14-reductase, stimulated the resumption of meiosis in cumulus-enclosed oocytes (CEOs) but not in denuded oocytes (DOs), and it also could not induce the maturation of DOs cocultured with their cumulus cells. Therefore, it has been suggested that the physical interaction between the oocyte and cumulus cells is critical for FF-MAS synthesis in cumulus cells [18].
In view of the biological significance of the suggested role of FF-MAS and similar sterols in meiosis, we have decided to examine sterol synthesis and accumulation in various follicular compartments and, at the same time, the resumption of meiosis. Significantly, most of the available data regarding the involvement of FF-MAS in meiosis were obtained using isolated oocytes in which the spontaneous resumption of meiosis was inhibited by hypoxanthine or other drugs affecting oocyte cAMP levels. Therefore, we also decided to use follicle-enclosed oocytes (FEOs) in which meiosis is dependent on hormone stimulation, thus more closely resembling the physiological situation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rats from the Department of Biological Regulation, Wistar-derived colony were provided with water and rat chow ad libitum and housed in air-conditioned rooms with a 14L:10D photoperiod. The experiments were carried out in accordance with the principles and guidelines for the use of laboratory animals and were approved by the Weizmann Institute of Science research animal committee. Immature rats were injected with eCG (10 IU) between 0900 and 0930 h on Days 23–24 of age to enhance multiple follicular development. The animals were killed 48–50 h after eCG injection by cervical dislocation, and preovulatory follicles or COCs were explanted for culture.
Culture Media and Inhibitors
Oocytes and follicles were cultured in Leibovitz L-15 medium (Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum (Sera-Lab, Crawley Down, England), penicillin (100 U/ml) and streptomycin (100 µg/ml; Gibco). 1-Methyl-3-isobutylxanthine (MIX; nonselective inhibitor of phosphodiesterase [PDE]; Sigma, St. Louis, MO), AY-9944 (gift from Dr. P. Benveniste, Institut de Biologie Moleculaire des Plantes, Strasbourg, France), FF-MAS (gift from Dr. U.M. Rose, Organon, Oss, The Netherlands), and Org9935 (specific PDE3 inhibitor; gift from Dr. U.M. Rose) were kept in stock solution in ethanol (10 mM). Ovine LH (gift from Dr. A.F. Parlow, National Hormone and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Disease [NIDDK], Torrance, CA) was prepared in stock solution of 1 mg/ml in PBS (Gibco). The stock solution was further diluted for culture with the medium.
In Vitro Cultures of Follicles and COCs for Examination of Germinal Vesicle Breakdown
All manipulations were performed under a dissecting microscope (Nikon, Tokyo, Japan). The meiotic status was examined by Nomarski interference microscopy (Carl Zeiss, Oberkochen, Germany). Oocytes showing nuclear membrane (germinal vesicle) or only intact nucleolus were classified as immature. Oocytes that did not show any nuclear structures were classified as mature (germinal vesicle breakdown [GVB]).
Preovulatory follicles were excised as previously described [19]. The follicles (n = 10–15) were cultured for the indicated intervals either alone, with LH (100 ng/ml), with AY-9944 (100 µM), or in a combination of LH or AY-9944 and MIX (200 µM). At the end of culture, FEOs were released and collected for examination by pricking and gently pressing the follicles.
The COCs were collected into a medium containing MIX (200 µM) by puncturing the largest ovarian follicles with a 27-gauge needle and exerting gentle pressure. Before transfer to the test medium, the oocytes were washed twice in plain medium. Between 30 and 60 COCs were cultured in organ-culture dishes (Falcon, Cockeysville, MD) for 24 h in 1 ml of control or test media, as indicated in Results. Oocytes were pooled and distributed into at least three different treatment dishes. Each treatment was tested in at least three replicate cultures repeated in two separate experiments.
In Vitro Cultures of Follicles, COCs, and DOs for High-Performance Liquid Chromatography of Sterols
Follicles were cultured in the presence of 250 µCi R-[5-3H]mevalonate or 300 µCi [3H]acetate (Perkin-Elmer Life Sciences, Boston, MA) for 8 h either alone (control), with LH (100 ng/ml), with AY-9944 (100 µM), or with both LH and AY-9944. At the end of culture, the follicles were collected and frozen at –20°C for sterol extraction. Between 50 and 100 follicles were collected for each extraction.
The COCs (n = 200–300 per dish) were cultured for 8 h either alone (control), with LH (100 ng/ml), or with AY-9944 (10 µM) in presence of 162 µCi [3H]mevalonate or 600 µCi [3H]acetate. At the end of culture, the COCs were collected with the medium and frozen at –20°C for sterol extraction. Between 400 and 700 COCs were collected for each extraction.
The DOs were recovered from COCs. Cumulus cells were removed by incubation with a mild chelating agent (3 mM EDTA [pH 8.0] in L-15 at 37°C for 30 min), followed by repetitive pipetting. The DOs (n = 200– 400 per dish) were cultured for 8 h either alone (control) or with AY-9944 (10 µM) in presence of 125 µCi [3H]mevalonate or 600 µCi [3H]acetate. At the end of culture, the DOs were collected with the medium and frozen at –20°C for sterol extraction. Between 900 and 1500 DOs were collected for each extraction.
Sterol Extraction from Follicles, COCs, and DOs
For follicles, identical frozen samples were combined and homogenized in 100 µl of sucrose-Tris buffer (0.25 M sucrose and 10 mM Tris [pH 7.4]) with five strokes of the pestle at 30% of full speed. Sucrose-Tris (100 µl) was used to rinse the pestle and was added to raise the final volume of 200 µl. For COCs and DOs, the samples were frozen and thawed five times in a dry-ice bath before saponification.
Saponification was carried out by adding 1.5 volumes of 7.5% KOH in 90% EtOH and heating at 70°C for 2 h. The samples were then transferred from Eppendorf tubes to glass tubes, and the Eppendorf tubes were rinsed with 100 µl of H2O.
To quantify the recovered sterols, 10 µg each of FF-MAS (gift from Dr. Christian Grøndahl, Novo Nordisk A/S, Denmark), cholesterol, progesterone, and 7 µg of lanosterol were added to each sample. The aqueous solutions were each extracted four times for 1 min with 2 volumes of water-saturated hexane (high-performance liquid chromatographic [HPLC] reagents; Biolab, Israel). The hexane layers were combined and evaporated to dryness under N2. The dried residues were stored at –20°C for analysis by HPLC.
HPLC Analyses
The HPLC analyses were performed on a Hewlett-Packard HPLC system (series 1050; Hewlett-Packard, Germany). The sterol samples were reconstituted in 50 µl of heptane and were loaded onto a straight-phase column (length, 250 mm; inner diameter, 4.6 mm; film thickness, 5 µm; ChromSpher Si; Varian), running in heptane/0.5% isopropanol at 1 ml/min [18]. The sterols were detected by ultraviolet absorption and were identified by their characteristic absorption spectra between 200 and 300 nm. Lanosterol and FF-MAS overlapped and were eluted between 9 and 12 min, cholesterol at approximately 21 min, and progesterone at approximately 36 min.
The lanosterol plus FF-MAS fraction, cholesterol, and progesterone were each evaporated to dryness and stored at –20°C until the next step. Further purification was achieved on a silver column prepared according to the method described by Ruan et al. [20] and was eluted with hexane/ 9.1% acetone. Lanosterol and FF-MAS were separated with an initial elution at 1 ml/min, which brought off lanosterol at approximately 11–12 min. After 15 min, the rate of elution was increased to 1.7 ml/min, which bring off MAS after approximately 60 min. Cholesterol and progesterone were chromatographed separately, with cholesterol eluting at approximately 23–24 min and progesterone at approximately 26 min. All samples were evaporated to a volume of between 1 and 2 ml and were stored at –20°C. The quantity of each compound was determined from the area under the curve of the compound eluted from the silver column compared with the average area found from running several standard amounts of each compound. The radioactivity in each compound was determined by counting an aliquot from each sample in a liquid scintillation counter.
Thin-layer chromatography (TLC) of each sample was carried out (Silica gel 60 F254; Merck, Germany) using an eluting solution of hexane/ 40% ethyl acetate [11] to verify the radiochemical purity. Usually, approximately 500–1000 counts of each compound were applied to the TLC plate along with standards. After development, the plates were dried in air, and the spots were visualized under ultraviolet light and by exposure to I2 vapor. A 1-cm area around the spot belonging to the compound was scraped off, and the remainder of the lane was divided into four rectangular areas and then scraped off as well. All scrapings were placed in counting vials along with scintillator fluid. In most cases, 90% or more of the total radioactivity on the plate was recovered in the spot belonging to the particular sterol. If the purity was less than 90%, the calculated amount of the compound synthesized was corrected.
Calculation of Sterol Synthesized
The calculation of sterol synthesized [21] depends on the assumption that the specific activity of the synthesized sterol is a function of the specific activity of the radioactive substrate, because virtually all the synthesized compound comes from the radioactive compound and not from an endogenous source. In the case of R-[5-3H]mevalonate, six molecules enter the sterol ring. Therefore, the specific activity of a mole of sterol is sixfold the specific activity of the mevalonate, whereas progesterone incorporates five mevalonate molecules. In the case of [3H]acetate, an inspection of the pathway shows that a mole of cholesterol contains 19 [3H]atoms, a mole of lanosterol 26 [3H]atoms, a mole of FF-MAS 24 [3H]atoms, and a mole of progesterone 14 [3H]atoms. Thus, for example, the specific activity of synthesized lanosterol is 26-fold the specific activity of each [3H]. The specific activity of each [3H] is one-third the specific activity of acetate.
If M0 is the amount of cold compound added, SAr the specific activity of the pure compound reisolated from the incubation mixture, SAu the specific activity of the compound synthesized, and Mu the amount of compound synthesized, then
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Statistical Analysis
Statistical analysis by ANOVA and Student t-test was performed when appropriate. Multiple samples were compared using the Fisher least squares difference procedure. Values of P < 0.05 were considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In follicles, both LH and AY-9944 markedly increased lanosterol and FF-MAS accumulation (Fig. 1, A and B). At the same time, as expected, AY-9944 suppressed cholesterol synthesis even more effectively (95–97%) (Fig. 1C). Furthermore, LH and AY-9944 had additive effects only on FF-MAS accumulation (Fig. 1B). Progesterone synthesis from mevalonate or acetate was not detected in follicles; therefore, it was not purified in further studies on COCs and DOs (data not shown). This may relate to the limited contribution of de novo cholesterol synthesis in steroidogenic tissues that are rich in cholesteryl esters, such as preovulatory follicles [22].
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In COCs, basal lanosterol production was relatively higher than in follicles (approximately 4- to 10-fold) (Figs. 1A and 2A). Basal and LH-stimulated FF-MAS synthesis in COCs was similar to that in follicles (Figs. 1B and 2B). The FF-MAS accumulation was enhanced effectively by AY-9944 (Fig. 2B), but LH had minimal or no effect on either lanosterol or cholesterol synthesis (Fig. 2, A and C). Like in follicles, AY-9944 dramatically decreased cholesterol synthesis in COCs (Fig. 2C).
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In DOs, lanosterol synthesis was detected both in the control and in the AY-9944-stimulated group (Fig. 3). Both cholesterol and FF-MAS were detected in trace amounts (0.5 and 4 fg/µg protein, respectively) in only the mevalonate control group (data not shown). Therefore, we did not repeat these experiments to save animals and labor.
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Effects of Endogenous FF-MAS Accumulation on FEO or CEO Maturation
Both LH and AY-9944 were able to stimulate resumption of meiosis in rat FEOs after 8 or 12 h of culture (Fig. 4) when FF-MAS accumulation was already increased (Fig. 1B). The stimulation of meiosis was blocked by MIX (a nonselective inhibitor of PDEs). In addition, MIX caused remarkable degeneration of FEOs treated by AY-9944 after 12 h of culture.
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Consistent with the results obtained in the FEO cultures, AY-9944 (10 µM) could not overcome the inhibition of spontaneous CEO maturation by MIX (100 µM) after 24 h of culture (Fig. 5), even though it had increased FF-MAS accumulation dramatically after 8 h of culture (Fig. 2B). A higher dose of AY-9944 (25 µM) also could not increase CEO maturation after 6 or 12 h of culture; instead, it markedly increased degeneration after 12 h of culture (data not shown).
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Effect of Exogenous FF-MAS on CEO Maturation
As shown in Figure 6, the spontaneous CEO maturation was prevented by either MIX (100 µM) or Org9935 (0.5 µM), a specific inhibitor of PDE3 that is expressed in the ovary only in the oocytes [23, 24]. Addition of an effective dose of FF-MAS (10 µM) did not increase the maturation rate in the presence of any of these two PDE inhibitors. Similarly, FF-MAS did not induce maturation of rat DOs cultured with MIX (data not shown).
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Timing of Meiotic Resumption Induced in FEOs by LH or AY-9944
Resumption of meiosis induced in rat FEOs by the addition of AY-9944 (100 µM) reached its peak (87%) only after 8 h of culture, and further extension of the culture period led to an increase in the number of degenerating oocytes (Fig. 7, broken line). On the other hand, when meiosis was triggered by LH (100 ng/ml), meiotic maturation reached a plateau (84%) after 4 h of culture. Thus maturation of FEOs induced by AY-9944 was delayed approximately 4 h compared to that induced by LH.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In COCs, in contrast to follicles, LH had little or no effect on cholesterol synthesis, which might relate to the low number of LH receptors on cumulus cells compared to granulosa cells. Nevertheless, the synthesis of lanosterol in COCs is relatively higher than in follicles, which might indicate that lanosterol synthesis in COCs is more active than in the other follicular compartments. Again, FF-MAS accumulation was dramatically increased by AY-9944.
In DOs we were able to detect basal and AY-9944-stimulated de novo synthesis of only lanosterol. However the synthesis of both FF-MAS and cholesterol was generally less than the detectable level. By contrast, using immunohistochemical staining, we have observed a preponderance of the LDM stain in oocytes [17]. The discrepancy between the high LDM staining in oocytes and our inability to detect more than trace amounts of MAS or cholesterol in DOs may result from insufficient sensitivity of the method employed. Alternatively, this discrepancy may relate to a deficiency in LDM or other enzyme activity in the biosynthetic pathway to cholesterol. Thus, factors from other follicular compartments may be necessary for this activity. Also, it may be caused by an inhibitory action of MAS after stimulation with AY-9944.
Considering the protein content of the follicles, COCs, and DOs, the bulk of sterol synthesis occurs in other follicular compartments and not in the COCs. Thus, the stimulation of FF-MAS by gonadotropins occurs primarily in other follicular compartments and not only in cumulus cells.
In follicles we found an additive effect of LH and AY-9944 on MAS synthesis and accumulation. It might suggest that MAS synthesis stimulated by LH is caused by activation of an upstream rate-limiting enzyme for sterol synthesis. Our previous studies have shown that mRNA expression of LDM is upregulated by hCG and that the elevation of its protein appears in COCs only after 6 h [17]. In addition, LH decreases cholesterol synthesis, which may be related to FF-MAS accumulation. In rat testis, the lack of a coordinate transcriptional control over the cholesterol biosynthetic pathway (3-hydroxy-3methylglutaryl-coenzyme A [HMG-CoA] synthase, HMG-CoA reductase, farnesyl diphosphate synthase, squalene synthase, and LDM were upregulated, whereas 4-demethylase and 7-reductase were at low level and not upregulated) contributes to overproduction of T-MAS. This control seems to be independent of sterol regulatory element-binding protein but dependent on cAMP/cAMP response element modulator [26]. However, in rat testis and hepatic HepG2 cells, progestins were shown to inhibit 24-reductase and 4-demethylase, resulting in MAS accumulation [27]. Clearly, further studies concerning the regulation of the enzymes involved in cholesterol synthesis are required to clarify the mechanism of FF-MAS accumulation by LH.
Both LH and AY-9944 increased FF-MAS accumulation dramatically in follicles, and AY-9944 did the same in COCs. This increase of FF-MAS did not alter the inhibitory action of MIX on meiosis. The addition of FF-MAS to the culture medium also could not increase COC maturation in the presence of PDE inhibitors, such as MIX and Org9935. Thus, our data suggest that neither endogenous FF-MAS accumulation nor exogenous addition of FF-MAS can overcome the inhibition of oocyte maturation by PDE inhibitors. By contrast, Hegele-Hartung et al. [28] reported that MAS, but not LH, overcame the inhibition of meiosis both in vitro and ex vivo. Such discrepancy in the action of LH and FF-MAS is difficult to reconcile with the suggested role of FF-MAS as a transducer of LH action on the resumption of meiosis.
Comparing the time sequence of AY-9944-induced maturation in rat FEOs with that stimulated by LH reveals a marked delay. In the other two models examined, mouse and rat CEOs and DOs, addition of FF-MAS also resulted in delayed maturation [13, 28, 29]. Furthermore, in mouse ovaries, the concentration of MAS did not reach detectable levels until after GVB had already been observed [25]. Collectively, the delayed meiotic response to FF-MAS stimulation and the FF-MAS induction preceded by GVB in response to LH make it unlikely that MAS serves as an obligatory downstream mediator of LH action on meiosis. Nevertheless, the upregulation of MAS by gonadotropins allows it to exert other beneficial actions on the follicle/ oocyte rather than induction of the resumption of meiosis. Recently, it has been demonstrated that FF-MAS improved oocyte quality, as shown by the progression from metaphase I to metaphase II and the increase in developmental competence of mouse oocytes [30]. The exact functions of MAS, as well as its precise mode of action, need further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: FAX: 972 8 9344116; alex.tsafriri{at}weizmann.ac.il
Received: 10 May 2004.
First decision: 28 May 2004.
Accepted: 15 July 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-demethylation of lanosterol by yeast. Biochem Biophys Res Commun 1986 134:659-663[CrossRef][Medline]-demethylase during the preovulatory period. Biol Reprod 2001 64:299-30914-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro. J Reprod Fertil 2000 118:171-179This article has been cited by other articles:
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  C.-G. Liang, Y.-Q. Su, H.-Y. Fan, H. Schatten, and Q.-Y. Sun Mechanisms Regulating Oocyte Meiotic Resumption: Roles of Mitogen-Activated Protein Kinase Mol. Endocrinol., September 1, 2007; 21(9): 2037 - 2055. [Abstract] [Full Text] [PDF]
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 S. Cukurcam, I. Betzendahl, G. Michel, E. Vogt, C. Hegele-Hartung, B. Lindenthal, and U. Eichenlaub-Ritter Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes Hum. Reprod., March 1, 2007; 22(3): 815 - 828. [Abstract] [Full Text] [PDF]
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