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Regular Article |
-Demethylase During the Preovulatory Period1
a The Bernhard Zondek Hormone Research Laboratory, Department of Biological Regulation, the Weizmann Institute of Science, Rehovot 76100, Israel
ABSTRACT
In vitro studies on mouse oocytes have shown that two closely related sterols, subsequently named meiosis-activating sterols (MAS), can overcome the inhibitory effect of hypoxanthine on the resumption of meiosis. These sterols are synthesized by cytochrome P450 lanosterol 14
-demethylase (LDM), a key enzyme in cholesterol biosynthesis. We have used specific inhibitors of LDM, azalanstat (RS-21607) and RS-21745, to test whether MAS is an obligatory mediator in the resumption of meiosis in the rat. Addition of azalanstat and RS-21745 (1–200 µM) to culture medium of rat isolated cumulus-enclosed oocyte and preovulatory follicle-enclosed oocyte stimulated by LH/hCG did not allow separation between their inhibition of the resumption of meiosis and the degeneration of oocytes. In both models, doses of the drug that inhibited oocyte maturation also increased oocyte degeneration. The inhibitors only partially suppressed follicular progesterone production. We have examined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunocytochemistry the ovarian expression of LDM mRNA and protein during the preovulatory period. We did not find evidence for the stimulation of this enzyme by LH/hCG. The strongest staining by LDM antiserum was obtained in primordial and primary oocytes, and the staining was reduced with oocyte growth. In addition, strong LDM staining could be observed in some of the granulosa cells, especially of the corona radiata localized in close proximity to the oocyte. In conclusion, our results with specific inhibitors and molecular approaches do not reveal evidence to support the hypothesis that MAS is an obligatory step in the stimulation of the resumption of meiosis. Specific inhibitors of MAS synthesis did not prevent spontaneous or LH-stimulated meiosis at doses that have previously been shown to effectively suppress LDM activity. Much higher concentrations of the inhibitors, which affected meiosis, were detrimental to oocytes, leading to their degeneration. The timing of LDM expression in the ovary was incompatible with a role for MAS in meiosis. Finally, the preferential localization of LDM protein to the oocytes suggests MAS production in oocytes rather than its transport from the somatic compartment as implied by the proposed role of MAS as a cumulus-oocyte signal molecule.
cumulus cells, follicle, gamete biology, meiosis, oocyte development, ovum
INTRODUCTION
Culture of mouse isolated oocytes with inhibitors of spontaneous resumption of meiosis, such as hypoxanthine, suggested that a positive regulator produced by somatic cells is transported to the oocyte and induces its maturation [1–4]. Using the same model system, Byskov et al. [5] identified this putative regulator as meiosis-activating sterols (MAS) in human follicular fluid (FF-MAS) and bull testicular tissue (T-MAS). These two sterols (4,4-dimethyl-5-cholest-8,14,24,-triene-3-ol from follicular fluid and 4,4-dimethyl-5-cholest-8,24,-diene-3ß-ol from bull testes) as well as two closely related synthetic C29 sterols modestly increased the proportion of maturing mouse oocytes cultured with hypoxanthine as an inhibitor. The ability of MAS and several of its derivatives to cause resumption of meiosis in the presence of hypoxanthine was confirmed by additional laboratories [6, 7].
A key enzyme in cholesterol biosynthesis is lanosterol 14-demethylase (LDM), which is encoded by the CYP51 gene [8–10], a member of the cytochrome P450 (CYP) gene superfamily involved with sterol synthesis in fungi, plants, and animals [11]. The human and rat LDM are 93% identical [9]. The FF-MAS is formed by LDM-dependent demethylation of lanosterol and converted to T-MAS by a sterol 14-reductase [12, 13]. The enzyme LDM is stimulated in immature rat ovaries by eCG, resulting in a sixfold increase within 48 h, and the activity of ovarian and testicular LDM is inhibited in vitro by ketoconazole [14]. By the use of ketoconazole, we have failed to confirm the suggested role of MAS in the resumption of meiosis in the rat, both in vivo or in vitro [15]. Therefore, using well-characterized rat models, we have tested the actions of the potent and highly specific LDM inhibitors, azalanstat (RS-21607) and RS-21745, with a median effective dose (ID50) in the nanomolar range [16, 17] on the resumption of oocyte maturation in vitro. It was assumed that if the C29 sterols play a physiological and essential role in the resumption of meiosis and, thus, function as MAS, azalanstat and similar inhibitors should block or attenuate the spontaneous maturation of isolated oocytes and of follicle-enclosed oocytes stimulated to mature by LH/hCG. The drug AY9944 was previously shown to block sterol-
7-reductase [18] and sterol-14-reductase [13]. The latter activity resulted in the accumulation of FF-MAS and also in germinal vesicle breakdown (GVBD) of hypoxanthine-blocked mouse oocytes [19]. Therefore, we have tested the effects of AY9944 on the maturation of rat oocytes.
The suggested role of MAS in mediation of the action of LH/hCG on the resumption of meiosis was also examined by the temporal and spatial expression of LDM mRNA and protein as revealed by polymerase chain reaction (PCR), Western blotting, and immunocytochemistry of ovarian tissues.
MATERIALS AND METHODS
Animals
Rats from the Department of Hormone Research Wistar-derived colony were provided with water and rat chow ad libitum and housed in air-conditioned rooms illuminated for 14 h/day. The experiments were carried out in accordance with the principles and guidelines for the use of laboratory animals and approved by the Weizmann Institute of Science research animal committee. Immature rats were injected with eCG (10 IU) between 0900–0930 h on Days 23–24 of age to enhance multiple follicular development. For testing ovulation in vivo, the animals were stimulated to ovulate by hCG (4 IU) 53 h later (afternoon of "proestrus"). For explanting cumulus-enclosed oocytes (CEOs) or follicle-enclosed oocytes (FEOs) for culture, the animals were killed 48–50 h after eCG by cervical dislocation.
Ovulatory Response In Vivo
Two days after eCG treatment, immature rats were administered the indicated doses of azalanstat or RS-21745 or vehicle into their periovarian sac (bursa) between 1230–1330 h. For intrabursal injection, the animals were anesthetized by a cocktail of ketamine (40–60 mg/kg) and diazepam (2–3 mg/kg), and one of the ovaries was exteriorized via a small lumbosacral incision. A 29-G needle was threaded into the ovarian bursa via the adjoining fat pad. The location of the injection was confirmed by the observation of the swelling of the bursa. After injection of the inhibitor or vehicle (100 µl/bursa), the ovary was replaced into the abdominal cavity, and the skin was sutured. The contralateral ovary served as control. Our previous studies established that intrabursal injection of saline does not significantly affect the number of ovulated ova [20, 21].
Culture Media and Inhibitors
Oocytes and follicles were cultured in Leibovitz's 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). IBMX (Sigma, St. Louis, MO) was kept in stock solution in dimethyl sulfoxide (10 mM), and azalanstat (RS-21607), RS-21745 (gift of Dr. D.C. Swinney, Roche Bioscience, Palo Alto, CA), and AY9944 (gift of Dr. P. Benveniste, Institut de Biologie Moleculaire des Plantes, Strasbourg, France) were dissolved in ethanol. This stock solution was further diluted for culture with the medium. The same vehicle was either injected into control rats or included in control culture media. The animals were killed the next morning, after which the ampullae of the oviducts were excised and the ova released, counted under a dissecting microscope, and then transferred for evaluation of their meiotic status under Nomarski interference contrast microscopy (Zeiss, Oberkochen, Germany).
Oocyte Collection and Culture
All oocyte manipulations were performed under a dissecting microscope. Oocytes were collected into a medium containing IBMX (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. Twenty-five to fifty oocytes, within their attached cumulus cell mass (i.e., "isolated oocytes"), were cultured in organ-culture dishes (Falcon, Cockeysville, MD) for 6 or 24 h in 1 ml of control or test media, as indicated in Results. Oocytes from three rats 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.
Follicle Cultures
Immature Wistar-derived rats treated with eCG (10 IU) were killed on the morning of the day of proestrus by cervical dislocation. Preovulatory follicles were excised under a dissection microscope as previously described [22]. The follicles (5–10 per dish) were cultured for 24 h alone or with LH (5 µg/ml) or in a combination of LH and the indicated concentration of the inhibitors.
Examination of Oocytes
At the end of the 6- or 24-h culture of isolated oocytes, oocytes were collected for microscopic examination. Follicle-enclosed oocytes were released and collected after 24 h of culture by making a small incision in the follicle. Some of the ovulated oocytes from the bursae as well as oocytes retained in unruptured, large follicles were examined for their meiotic status by Nomarski interference microscopy. Oocytes showing clear nuclear membrane (germinal vesicle) or only intact nucleolus were classified as immature. Oocytes that did not show any nuclear structures, because they had undergone GVBD, were classified as mature [23].
Steroid RIA
The accumulation of progesterone and estrogen in medium from follicular cultures was measured by a previously described RIA procedure [24].
Reverse Transcriptase-PCR Analysis
The expression of LDM was examined by relative-semiquantitative reverse transcriptase (RT)-PCR as previously described [25]. Total RNA was extracted from ovaries, preovulatory follicles (10 per replicate), and oocyte cumulus complexes (150 per replicate) at the indicated time intervals, and treatments by the acid-guanidium-phenol-chloroform method [26] and aliquots of 50 ng of RNA from ovaries, 75 ng of RNA from preovulatory follicles, or total RNA from 150 oocyte-cumulus complexes (OCCs) were reverse-transcribed using random primers followed by PCR amplification. The RT reaction contained 50 U of Moloney murine leukemia virus-RT, 200 mM deoxynucleotide triphosphate (dNTP), 6.5 mM MgCl2, 20 U of RNAsin, 500 mg of oligo deoxythymidine, and 1.5x PCR buffer (Promega, Madison, WI). The reaction was performed at 37°C for 2 h. Fragments of the reverse-transcribed LDM cDNA were amplified using a labeled nucleotide (-32P-deoxycytidine triphosphate (dCTP); Amersham, Buckinghamshire, UK), and the following pair of primers was employed: 5'-GGTGGCTCAGCTGTACGCAGACCTGG-3', and 5'-ATTTGAACATAGGCAAAATTTTCTCC-3' [27]. A fragment of GAPDH cDNA, which served as an internal standard, was amplified in parallel using the following primers: 5'-GCCATCAACGACCCCTTCAT-3', and 5'-TTCA- CACCCATCACAAACAT-3'.
The PCR reactions were further performed in the same RT test tube that finally contained 250 ng of each primer, 200 mM dNTP, 2.5 mM MgCl2, 2 mCi -32P-dCTP, 1x PCR buffer (Promega), and 2.5 U Taq polymerase. Annealing temperature of 60 and 55°C for LDM and GAPDH during 28, 20, and 28 cycles for ovaries, follicles, and OCCs, respectively, were performed. The radioactive products were electrophoresed on 5% nondenaturing polyacrylamide gel in 0.5x Tris-borate-EDTA buffer. Gels were dried, and radioactivity was determined by exposing to x-ray film for 1 h at -70°C. Quantitation and comparison of the autoradiograms were performed by densitometric analysis (Quantity One; PDI, 420oe, New York, NY), and the expression level normalized to GAPDH.
Western Blot Analysis of LDM
Antibodies against the synthetic peptide H-Lys-Glu-Arg-Leu-Asp-Phe-Asn-Pro-Asp-Arg-Tyr-Leu-Gln-Asp-Asn-Pro-Ala-Ser-Gly-Glu-OH derived from the rat LDM sequence [27], corresponding to Glu419–Glu437, were raised in rabbits by s.c. injection of the peptide cross-linked to keyhole limpet hemocyanin as a carrier protein. A standard immunization protocol was utilized. Ovaries, preovulatory follicles, and OCCs were homogenized in RIPA buffer (20 mM Tris-HCl [pH 7.4], 137 mM NaCl, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100, 2 mM EDTA [pH 8.0], 1 mM PMSF, and 20 mM leupeptin) and resolved by 10% SDS-PAGE. The resolved protein bands were electrophoretically transferred from the gels onto nitrocellulose. The nitrocellulose blots were blocked with 10 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween 20 containing 2% BSA. The blots were treated with polyclonal rabbit LDM antiserum (1:2500). Staining was performed with Protein A (ICN, Costa Mesa, CA) conjugated to horseradish peroxidase (1:30 000) and using enhanced chemiluminescence light-generating reagents (Amersham). Quantitation and comparison of the bands were performed by densitometric analysis as described above, and the expression level was normalized to hCG 0-time.
Immunohistochemistry
Ovaries were explanted 48 h after eCG (10 IU) or 3, 6, and 9 h after hCG (4 IU) treatment, trimmed of fat, fixed overnight in Bouin's solution, and then processed to paraffin wax blocks. Sections were cut (5 mµ), mounted on poly-L-lysine-coated slides, dewaxed, hydrated, and processed using Histostain-SP kit (95–6143; Zymed Laboratories, San Francisco, CA) according to the manufacturer's instructions. All slides were incubated in peroxidase quenching solution (one part 30% hydrogen peroxide to nine parts absolute methanol) followed by serum blocking solution. Alternate slides were incubated with the primary antibody, anti-LDM, or the preimmune serum (1:200). This was followed by consecutive incubation with biotinylated second antibody, streptavidin-horseradish peroxidase conjugate, and substrate-chromogen mixture with hydrogen peroxide. The slides were counterstained with hematoxylin to visualize the tissue and mounted with GVA mounting solution (Zymed Laboratories). Preincubation of slides with the antigen as well as incubation with the preimmune serum instead of the anti-LDM prevented staining of the tissue.
Statistical Analysis
Statistical analysis by ANOVA and Student's t-test was performed whenever appropriate. Values of oocyte maturation SEM and statistical significance were calculated according to the method described by Ott [28]. Values of P < 0.05 were considered to be significant.
RESULTS
LDM Inhibitors and Resumption of Meiosis in CEOs
Addition of azalanstat (1–10 µM) or RS-21745 (10 µM) to the medium did not affect the spontaneous resumption of meiosis after 6 h of culture. Nevertheless, higher concentrations of the inhibitors (50–100 µM) or extension of the culture to 24 h (10–100 µM) reduced the percentage of mature oocytes that was associated with dose- and time-dependent increase in oocyte degeneration (Figs. 1 and 2). Up to 3 h of culture, azalanstat (1–50 µM) did not affect the rate of GVBD as compared to the control, but the highest dose tested (100 µM) significantly reduced the percentage of mature oocytes (31%) and resulted in degeneration (10%) of oocytes as compared to control cultures (data not shown). When CEOs were cultured with azalanstat for 6 h, washed, and then transferred into a fresh, inhibitor-free control medium for an additional 24 h, the inhibitory effect of the drug was partially reversed only in the 50 µM concentration, whereas the higher concentration of 100 µM caused more than 85% degeneration (Fig. 3).
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Effects of LDM Inhibitors on FEOs
Azalanstat and RS-21745 dose-dependently reduced the number of mature oocytes in FEO cultures due to an increase in the degeneration rate. This effect was more clearly seen in oocytes cultured for 24 h with the drug (Figs. 4 and 5). Both inhibitors reduced LH-stimulated progesterone accumulation in the medium, but RS-21745 was somewhat more potent (Fig. 6). This activity of azalanstat and RS-21745 was less effective than that of ketoconazole [15], and the residual progesterone accumulation was, in all cases of LH stimulation, greater than that of the unstimulated control. Likewise, when administered locally into the ovarian bursa, both inhibitors affected ovulation only marginally (in the dose range of 2.15–215 µg/bursa, only azalanstat at the highest dose resulted in significant [P < 0.05] inhibition of ovulation), whereas ketoconazole effectively inhibited ovulation [15].
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Effects of AY9944 on Oocyte Maturation
Addition of AY-9944, which inhibits the activity of 14-reductase and transformation of FF-MAS to T-MAS, to CEOs in culture did not affect the rate of GVBD during a 6-h culture period, but the highest dose (25 µM) increased the degeneration rate (Fig. 7). When added to FEOs, AY9944 (50–100 µM) caused the resumption of meiosis on its own, even without LH. The highest dose used as well as prolongation of the FEO cultures to 24 h revealed that AY9944 causes degeneration of oocytes in FEOs. This effect of the drug was also observed when it was given with LH (Fig. 8). The drug alone stimulated progesterone accumulation in the medium, and when combined with LH, it somewhat reduced progesterone accumulation during the 6-h culture (Fig. 9).
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Expression of LDM mRNA
Relative quantitative RT-PCR did not reveal an increase in ovarian LDM mRNA expression after eCG administration to immature rats (Fig. 10A); the relative rat LDM/GAPDH values were 1.17 ± 0.13, 1.00 ± 0.08, and 1.02 ± 0.18 at 0, 24, and 48 h, respectively, after eCG. By contrast, hCG treatment of these rats did cause an increase in ovarian (Fig. 10B), follicular (Fig. 10, C and D), and denuded oocyte mRNA (100%, 155%, and 244% at 0, 1.5, and 3 h, respectively after hCG injection; data not shown).
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Expression of LDM Protein
Western blot analysis revealed a 33% (P < 0.05) increase in ovarian expression of LDM 48 h after eCG stimulation, but hCG treatment of rats did not show a further significant increase in LDM protein expression (Fig. 11). Likewise, in explanted follicles, hCG injection in vivo (1–6 h) did not elicit an increase in LDM expression, and 9 h after the treatment, it was somewhat reduced (Fig. 12). In OCCs explanted at 1–3 h after hCG injection, no increase was seen in LDM expression as compared to 0-time, but an increase was noted at 6 (P < 0.05) and 9 (P < 0.0001) h after hCG stimulation (Fig. 13), a time point too late to assume that its product, MAS, is needed for mediation of the hCG stimulation of the resumption of meiosis.
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Immunohistochemistry revealed strong LDM expression in oocytes at all stages of follicular growth. The strongest staining was in small oocytes, and the deposit was somewhat reduced with oocyte and follicle growth. The oocytes were clearly the most heavily stained, with interstitial and theca cells, as well as granulosa cells, showed faint staining (Fig. 14). More detailed examination of earliest stages of follicular development showed that oocytes from primordial, transitional, and primary follicles were stained (Fig. 15). In some follicles, it was possible to locate a group of granulosa cells in close proximity of the oocyte showing considerably heavier staining than the rest of the granulosa cells (Fig. 16). Examination of ovaries from eCG- or from eCG- and hCG-treated immature rats (3, 6, or 9 h after hCG) did not reveal detectable differences in immunocytochemical staining of LDM. Therefore, the data shown in Figures 14 through 16 were all from rats treated only with eCG.
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DISCUSSION
Female gamete production is subject to several stop/go controls [29–31]. The oocytes initiate the first meiotic division during embryonic development, whereas the meiosis of male germ cells is delayed. Using a fetal gonad coculture assay, it was suggested that the ovary produces an inducer of meiosis, which initially was termed meiosis-inducing substance and later renamed meiosis-activating substance (MAS), and that the embryonic testis produces a meiosis-preventing substance [32]. Human follicular fluid was shown to exhibit MAS activity as detected by the fetal testis assay [33, 34]. In cultured mouse isolated oocytes, human follicular fluid and extracts of bull testicular tissue could reverse the inhibition of spontaneous maturation by hypoxanthine. This activity was identified as a sterol, and the acronym MAS transformed from "meiosis-activating substance" to "meiosis-activating sterol". The one from human follicular fluid was referred to as FF-MAS and that from extracts of bull testicular tissue was referred to as T-MAS. The FF-MAS is the product of LDM demethylation of lanosterol and is converted to T-MAS by a sterol-14-reductase [12, 13].
We used azalanstat and RS-21745, two highly specific inhibitors of LDM with an ID50 in the nanomolar range, that have minimal effects against other CYPs examined [16, 17]. Because these inhibitors, at the lowest doses shown in other systems to effectively inhibit LDM, did not affect spontaneous or LH-induced resumption of meiosis, it seems unlikely that the product of LDM is a necessary intermediate in the resumption of meiosis. At higher concentrations of azalanstat and RS-21745 the reduced maturation rate was accompanied by a concomitant increase in the rate of oocyte degeneration. This suggests that the effect on meiosis of these inhibitors at high concentrations is not necessarily due to the inhibition of MAS production but may be due to toxic effects of the drugs, or more precisely, due to the detrimental effects of reduced cholesterol levels in the oocytes and follicle cells.
Comparing the effects of ketoconazole to these of azalanstat and RS-21745 underlines the much higher specificity of the latter two. Ketoconazole inhibited follicular steroidogenesis most effectively, reducing the LH-stimulated progesterone accumulation below that of the unstimulated control. Furthermore, when administered in vivo locally into the periovarian bursa, ketoconazole markedly suppressed ovulation [15]. Azalanstat and RS-21745 affected follicular steroidogenesis and ovulation only marginally.
Leonardsen et al. [19], tested the effects of AY9944, which inhibits sterol-14-reductase, the enzyme that transforms FF-MAS into T-MAS [13]. AY9944 stimulated FF-MAS accumulation and resumption of meiosis in mouse CEOs in the presence of hypoxanthine. We have extended these studies to the rat, demonstrating that AY9944, on its own, caused the resumption of meiosis in FEOs during the 6-h culture. Nevertheless, extension of the culture of follicles to 24 h revealed maturing oocytes as well as a high percentage of degenerating ones. AY9944 did not affect the spontaneous maturation of CEOs, but the higher doses of the inhibitor resulted in an increased degeneration rate. A similar effect of high AY9944 concentrations on oocyte degeneration has been observed in mouse oocytes as well [19]. In the rat, the drug also modified follicular steroidogenesis, similar to that occurring during follicular atresia [35]. In view of the degenerative actions of the drug in oocytes and the modification of follicular steroidogenesis, it is unsound to ascribe the meiosis-stimulating action of AY9944 solely to the accumulation of FF-MAS due to the inhibition of 14-reductase. The drug may also exert atretogenic effects on follicular somatic cells and oocytes.
The inhibitors used in this study were well characterized in several systems, both in vivo and in vitro [13, 16–18], and it is reasonable to assume that they exerted similar effects in our experimental setup. Yet, because we did not measure directly the effects of the inhibitors on MAS production and release in CEO and FEO cultures, our conclusions remain to be examined by direct determination of MAS synthesis and release in the presence of these inhibitors.
To act as a mediator of LH/hCG stimulation of the resumption of meiosis, according to the mechanism proposed by Byskov et al. [1, 36] and Leonardsen et al. [19], MAS should be produced in the somatic compartment of the follicle and transmitted to the oocyte after the gonadotropic surge. We have analyzed the temporal and spatial expression of LDM mRNA and protein using molecular and immunochemical approaches, and RT-PCR revealed an increase in LDM transcription only in response to hCG, not to eCG, treatment of immature rats. This increase in LDM mRNA expression was confirmed in whole-ovary, preovulatory follicle, and denuded oocyte preparations. Protein expression, as examined by Western blotting, revealed a different sequence of events. Stimulation with eCG increased ovarian LDM protein expression, but hCG had no marked effect in the whole ovary or preovulatory follicles. Only in the OCCs did hCG treatment increase LDM protein expression, but at a time (6 and 9 h after the stimulus) that was too late to be relevant to the resumption of meiosis.
Treatment of immature rats with eCG resulted in increased ovarian LDM activity and MAS sterol levels within 48 h [14]. The 50-fold increase in enzyme activity [14] and the meager 30% increase in protein level detected here, however, seem to suggest that ovarian LDM is regulated not only at the level of translation but also at the level of activity. Given the different methods employed, the regulation of LDM activity remains to be examined. The marked increase in LDM activity and ovarian FF-MAS after eCG stimulation, a time at which oocyte maturation (i.e., GVBD) does not occur and is pending upon an additional stimulus (i.e., exposure to endogenous or exogenous LH/hCG) [29, 31], argues against function of the sterol as the inducer of meiosis. If FF-MAS is the physiological trigger for the resumption of meiosis, its accumulation should set the process into motion even before the gonadotropin surge.
Immunocytochemistry localized the LDM enzyme primarily to the oocytes. The staining was heaviest in oocytes of primordial and primary follicles and was reduced with oocyte growth. Such distribution of LDM is incompatible with the suggested role of its product, MAS, as a trigger for the resumption of meiosis transmitted from the somatic compartment to the oocyte [19, 36]. The localization of LDM to oocytes is consistent with the elevated expression of LDM and synthesis of MAS in postmeiotic germ cells of male rats [37, 38]. The role of LDM products in germ cells remains to be determined. In the oocyte, it seems to be reasonable that the massive growth and assembly of membranous structures requires high synthesis of cholesterol, especially in growing oocytes. This may explain the heavier staining of small oocytes. The degenerative actions of all inhibitors of cholesterol synthesis on oocytes [15, 19] underlines the important role of cholesterol, and probably of other sterols, for oocyte well-being.
In naked mouse oocytes stimulated to mature by FF-MAS, GVBD was delayed when the spontaneous maturation was blocked by forskolin or hypoxanthine (7 and 11 h, respectively) [39]. Collectively, the inhibitor studies, the temporal and spatial expression of LDM, and the delayed GVBD when stimulated by MAS argue against the putative role of MAS as a physiological mediator of the gonadotropic stimulation of the resumption of meiosis. The precise mechanism (or mechanisms) by which MAS induces the resumption of meiosis, the regulation of follicular sterol synthesis, and their role in follicle and oocyte development and the ovulatory response remain to be thoroughly examined.
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ACKNOWLEDGMENTS
We thank Mrs. A. Tsafriri for her excellent technical help; Dr. A.F. Parlow and the NIDDKS National Hormone and Pituitary Program, NICHD, for the gonadotropins; Dr. D.C. Swinney (Roche Bioscience, Palo Alto, CA) for the azalanstat (RS-21607) and RS-21745; and Dr. P. Benveniste (Institut de Biologie Moleculaire des Plantes, Strasbourg, France) for the AY9944.
FOOTNOTES
1 Supported by the Maria and Bernhard Zondek Hormone Research Fund and by the Chief Scientist of the Ministry of Health, Jerusalem, Israel. A.T. is the incumbent of the Hermann and Lilly Schilling Foundation Professorship. 
2 Correspondence. FAX: 972 8 9344 116; alex.tsafriri{at}weizmann.ac.il
Accepted: August 28, 2000.
Received: June 5, 2000.
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X. Cao, S. H. Pomerantz, M. Popliker, and A. Tsafriri Meiosis-Activating Sterol Synthesis in Rat Preovulatory Follicle: Is It Involved in Resumption of Meiosis? Biol Reprod, December 1, 2004; 71(6): 1807 - 1812. [Abstract] [Full Text] [PDF]
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C. L. Marin Bivens, C. Grondahl, A. Murray, T. Blume, Y.-Q. Su, and J. J. Eppig Meiosis-Activating Sterol Promotes the Metaphase I to Metaphase II Transition and Preimplantation Developmental Competence of Mouse Oocytes Maturing in Vitro Biol Reprod, May 1, 2004; 70(5): 1458 - 1464. [Abstract] [Full Text] [PDF]
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Y. Yamashita, M. Shimada, T. Okazaki, T. Maeda, and T. Terada Production of Progesterone from De Novo-Synthesized Cholesterol in Cumulus Cells and Its Physiological Role During Meiotic Resumption of Porcine Oocytes Biol Reprod, April 1, 2003; 68(4): 1193 - 1198. [Abstract] [Full Text] [PDF]
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C. Grondahl, J. Breinholt, P. Wahl, A. Murray, T. H. Hansen, I. Faerge, C. E. Stidsen, K. Raun, and C. Hegele-Hartung Physiology of meiosis-activating sterol: endogenous formation and mode of action Hum. Reprod., January 1, 2003; 18(1): 122 - 129. [Abstract] [Full Text] [PDF]
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M. Baltsen Gonadotropin-Induced Accumulation of 4,4-Dimethylsterols in Mouse Ovaries and Its Temporal Relation to Meiosis Biol Reprod, December 1, 2001; 65(6): 1743 - 1750. [Abstract] [Full Text] [PDF]
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Y.-Q. Su, S. Rubinstein, A. Luria, Y. Lax, and H. Breitbart Involvement of MEK-Mitogen-Activated Protein Kinase Pathway in Follicle-Stimulating Hormone-Induced but Not Spontaneous Meiotic Resumption of Mouse Oocytes Biol Reprod, August 1, 2001; 65(2): 358 - 365. [Abstract] [Full Text] [PDF]
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