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a Research Laboratories of Schering AG, Berlin, Germany
b Health Care Discovery of Novo Nordisk A/S, Copenhagen, Denmark
c Department of Obstetrics and Gynaecology, Göteborg University, Sahlgrenska University Hospital, Göteborg, S-41345, Sweden
ABSTRACT
Meiosis-activating sterols (MAS) have been found to induce meiotic maturation in mouse oocytes in vitro. In the present study we have extended these observations by investigating the effects of follicular fluid MAS (FF-MAS) on rat oocyte maturation in vitro and ex vivo. Rat oocytes freed from their follicles were cultured with FF-MAS (0 µM, 1 µM, 3 µM, 10 µM, 30 µM) for 22 h in a medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 250 µM). A dose-dependent significant increase in germinal vesicle breakdown (GVB) was observed after adding FF-MAS to the culture medium in both cumulus-enclosed (CEO) and denuded (DO) oocytes. A time course study (0, 3, 8, 14, and 22 h) showed a significant increase in GVB after 14 h when DO and CEO were cultured in the presence of 10 µM FF-MAS + 250 µM IBMX. Furthermore immature rats were primed with eCG (20 IU) and 48 h later perfused ex vivo for 12 h in a recirculating system with either FF-MAS (0 µM, 10 µM, 30 µM, 60 µM), cholesterol (60 µM), or LH (0.2 µg/ml) in the presence of 200 µM IBMX, respectively. In addition, ovarian perfusion was carried out with FF-MAS (30 µM, 60 µM) or 0.2 µg/ml LH in the absence of IBMX. After 12 h, oocytes were freed from the ovaries and checked for GVB. By using the ex vivo perfused rat ovary, we found that FF-MAS, starting at 30 µM, was dose-dependently able to overcome IBMX-induced meiotic arrest leading to a comparable increase in GVB as was observed for LH. Furthermore, it was found that FF-MAS in the absence of IBMX was also able to induce meiotic maturation. Our data are consistent with the notion that the maturation-inducing effects of FF-MAS are mediated by different mechanisms compared to spontaneous maturation.
LH, meiosis, ovary, ovum
INTRODUCTION
Mammalian oocyte meiosis is initiated during fetal life and arrested at the dictyate stage of the first meiotic prophase I. The preovulatory LH surge leads to the resumption of oocyte meiosis in large preovulatory follicles in vivo. Oocytes, however, undergo a gonadotropin-independent meiotic maturation when removed from their ovarian follicles and are subsequently cultured in vitro [1]. Human oocytes, when removed from their follicular environment, also rapidly resume meiotic division [2]. This phenomenon is generally referred to as spontaneous meiotic maturation in vitro. Obviously, the follicular environment exerts an inhibitory effect on spontaneous oocyte maturation as demonstrated by decreased spontaneous maturation of cocultured granulosa cells [3] or by follicular fluid from several species [4].
Cyclic AMP is central to the regulation of meiosis. Several lines of evidence suggest that cAMP has an important role in maintaining the meiotic arrest of the oocyte. Spontaneous oocyte maturation can be prevented by compounds that maintain elevated cAMP levels [5], whereas meiotic maturation is resumed when the intracellular concentrations of cAMP decline [6]. The resumption of meiosis can be prevented by maintaining elevated cAMP concentrations in the oocyte. This can be achieved either by the inhibition of its phosphodiesterase (PDE)-associated degradation (e.g., 3-isobutyl-1-methylxanthine [IBMX]), or by the incorporation of exogenous cyclic nucleotides (e.g., dibutyryl cAMP) into the oocyte [7].
The mechanism by which LH induces resumption of meiosis in vivo remains a matter of controversy. There are two models that have been put forward to offer an explanation of the resumption of meiosis: 1) Meiotic maturation may result from the withdrawal of inhibitory signals to the oocyte and coincidentally, from the termination of the metabolic and cellular (e.g., gap junctions) coupling between the oocyte and granulosa cells [8]. Hence, breakdown of coupling between the oocyte and the granulosa cells may lead to an interruption of the cAMP transfer or of other inhibitory molecules to the oocyte. 2) Conversely, instead of depriving the oocyte of inhibitory substances, gonadotropins could induce maturation by a mechanism that bypasses the meiosis-arresting pathway. This process involves a positive factor produced by the follicular environment that acts on the oocyte to trigger the resumption of meiosis despite the continued presence of an inhibitor [9].
Substances exerting inhibitory effects on meiotic oocyte maturation, presumably the purine base hypoxanthine (Hx) and the nucleoside adenosine, have been shown to be present in porcine [10] and mouse [11] follicular fluids. In fact, these purines are present in high molar concentrations within follicular fluid and are capable of preventing meiotic resumption in oocytes from mice, rats, cows, and monkeys in vitro [11, 12]. Purines, such as Hx and IBMX, seem to maintain the oocyte meiotic arrest by modulating intracytoplasmic cAMP metabolism and also by modulating adenylate cyclase activity of the oocyte plasma membrane [13].
Recently, a C-29 lipid was discovered in the microenvironment surrounding the oocyte in the human follicular fluid [14]. This lipophilic molecule was shown to be a sterol (4,4-dimethyl-5
-cholesta-8,14,24-trien-3ß-ol) occurring naturally in the biosynthetic pathway between lanosterol and cholesterol. Contrary to lanosterol and cholesterol, this extracted sterol was shown to induce meiotic resumption in mouse oocytes in vitro and was thus defined as FF-MAS (follicular fluid meiosis-activating sterol). In accord with the meiosis-promoting effect of extracted FF-MAS, Grøndahl et al. [15] and Hegele-Hartung et al. [16] were able to show that synthetic FF-MAS is dose dependently able to trigger the resumption of meiosis in vitro in both denuded and cumulus-enclosed mouse oocytes arrested in meiosis with forskolin, Hx, IBMX, and dibutyryl cAMP. In addition, FF-MAS improved oocyte quality by supporting microtubule assembly and by delaying the cortical granule release [16] in mice.
The rat is a frequently employed animal model for studying ovarian physiology. However, the role for meiosis regulators has not been thoroughly investigated in this species, and nothing is known about the influence of FF-MAS on the resumption of meiosis. In the present communication, we were therefore interested in investigating the effect of FF-MAS on rat oocyte meiotic maturation in vitro. It is also still completely unknown whether FF-MAS, when administered into the circulation, is able to affect meiotic maturation in the oocyte in situ. The lipophilicity of sterols makes them often prone to deactivation in the body [17]. Therefore further studies in well-characterized models of ovarian physiology are needed to determine whether FF-MAS is able to trigger the resumption of meiosis in situ. In order to clarify whether FF-MAS, after administration into the ovarian circulation, is capable of triggering meiotic maturation, we used the ex vivo perfused rat ovary model [18].
MATERIALS AND METHODS
Animals
For the rat oocyte assay, immature female rats (Wistar Hannover, TZH; Schering AG, Berlin, Germany) weighing 45–55 g were used. Immature female Sprague-Dawley rats (Schönwalde, Berlin, Germany) with a body weight of 50–55 g were used for the ex vivo ovary perfusion. All animals were kept under controlled temperature (20–22°C), light (lights-on 0630–2030 h), and relative humidity (50–70%) with food and water ad libitum. All animal experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction and were run with the permission of the District Government of Berlin, Germany.
Source of FF-MAS
The C29-sterol 4,4-dimethyl-5-cholesta-8,14,24-trien-3ß-ol (FF-MAS) was synthesized by the Department of Medicinal Chemistry of Schering AG according to a synthesis described by Dolle et al. [19]. The purity of the finally described FF-MAS was checked and determined to be 99.3% by HPLC (column: Symmetry C18, 5 µ; column size: 150 x 3.9 mm; eluent: acetonitrile:water 95:5 containing 0.5 g/L ammonium acetate; detection: photodiode array at 214 nm).
Rat Oocyte Assay
Fully grown, germinal vesicle (GV)-intact oocytes were obtained from ovaries after priming with an s.c. injection of 20 IU eCG (AKZO Nobel, Oss, The Netherlands). Forty-eight hours after eCG injection, rats were killed by cervical dislocation. The ovaries were removed, freed of extraneous tissue, and placed in a multidish containing culture medium together with either 4 mM Hx (cat. no. H-9377; Sigma, St. Louis, MO) or 250 µM IBMX (cat. no. I-5879; Sigma) at 37°C. The culture medium used was -minimum essential medium (-MEM without ribonucleosides, cat. no. 22561; Gibco, Paisley, Scotland) supplemented with 8 mg/ml human serum albumin (HSA; State Serum Institute, Denmark), 0.23 mM pyruvate (cat. no. S-8636; Sigma), 2 mM glutamine (cat. no. 16-801; Flow Labs, McLean, VA), 100 IU/ml penicillin and 100 µg/ml streptomycin (cat. no. 16-700; Flow Labs).
The culture medium contained 4 mM Hx, designated as Hx-medium or 0, 5, 50, 250 µM IBMX, designated as IBMX-medium, to prevent GV breakdown (GVB). The oocytes were collected in either Hx- or IBMX-medium under a stereomicroscope by puncturing follicles using 27-gauge needles attached to a 1-ml syringe. Cumulus-enclosed oocyes (CEO) of uniform size were selected with a mouth-controlled pipette. Oocytes freed from cumulus cells, i.e., denuded oocytes (DO), were obtained by gently flushing CEO through a fine-bore mouth-controlled pipette. The CEO and DO were rinsed three times in Hx- or IBMX-medium and cultured without any oil in four-well multidishes (Nunclon, Wiesbaden, Germany) in which each well contained 0.4 ml of the respective medium and 30–50 oocytes. Only oocytes with an intact GV were used in the experiment.
The oocytes were cultured at 37°C in a humidified atmosphere of 5% CO2 in air. In the presence of 1) Hx (4 mM), 2) IBMX (5 µM, 50 µM, or 250 µM), and 3) FF-MAS (0 µM, 1 µM, 3 µM, 10 µM, or 30 µM FF-MAS in 250 µM IBMX-medium, respectively), DO and CEO were cultured for 22 h. In addition, DO and CEO were cultured for 0 h, 3 h, 8 h, 14 h, and 22 h with 250 µM IBMX, 10 µM FF-MAS + 250 µM IBMX, and without any FF-MAS and IBMX (spontaneously matured group).
By the end of the culture period, the number of oocytes with a GV, a GVB, and a polar body (PB), respectively, were counted using a stereomicroscope (Leica MZ 12; Wildt, Switzerland). The %GVB, defined as percentage of oocytes undergoing GVB per total number of oocytes, was calculated as: %GVB = (number of GVB + number PB/total number of oocytes) x 100.
For each experimental group a minimum of three experiments was conducted. Data were pooled and presented as mean ± SEM.
Ex Vivo Perfusion of the Rat Ovary
Rats were injected with 20 IU eCG (AKZO Nobel, Oss, The Netherlands) and 48 h later were anesthetized with an i.p. injection of ketamine (Ketavet; Pharmacia and Upjohn, Erlangen, Germany)-xylazine (Rompun; Bayer, Leverkusen, Germany) (105 and 8.3 mg/kg body weight, respectively). Three hundred IU of heparin sodium (cat. no. C-3045; Sigma) was injected into the tail vein. Ovaries with connecting vasculature were surgically isolated, and perfusions were performed in a recirculating system as previously described [20]. Briefly, the peritoneal cavity was opened and the distal colon resected, and all vessels connecting to the aorta and vena cava, except those of the right ovary, were ligated and severed. The aorta and vena cava were then cannulated in a retrograde direction and likewise ligated and severed above the branching of the renal vessels.
The excised ovaries, with bursa intact, were flushed with warmed (37°C) saline (0.9% NaCl; Fresenius, Homburg, Germany) and placed in the perfusion system containing 
40 ml of perfusion medium. The perfusion medium consisted of Medium 199 with Earles salts and L-glutamine without sodium bicarbonate (cat. no. 31100-027; Gibco) supplemented with 50 µg/ml gentamycin sulfate (cat. no. 15750-037; Gibco), 0.2 IU/ml insulin (Hoechst AG, Frankfurt, Germany), and 4% BSA (cat. no. 775835; Boehringer Mannheim, Germany). The perfusion medium was recirculated through the ovary for 1 h to allow metabolic stabilization of the tissue. Only the ovarian specimens that maintained a flow rate of 0.8–1.5 ml/min during this period, at a pressure of 80 mmHg, were used in the experiments.
After 1 h of preperfusion 0 (negative control), 10, 30, or 60 µM FF-MAS, respectively, 60 µM cholesterol (cat. no. C-8667; Sigma) or 0,2 µg/ml ovine LH (oLH with a specific activity of 20 IU; NIADDK, Bethesda, MD) were added to the perfusion medium in combination with 200 µM IBMX, respectively. In addition, 30 or 60 µM FF-MAS and 0.2 µg/ml oLH (positive control) were added to the perfusion medium without any IBMX. The IBMX and oLH were diluted in perfusion medium. For the investigation of meiosis, perfusions were continued up to 12 h after addition of the compounds. At the completion of the perfusion period, the ovaries were dissected free from adherent tissue and oocytes were isolated under a stereomicroscope by manual puncture of the follicles employing a pair of 27-gauge needles. The number of oocytes released after puncture per ovary was determined. The CEO were freed from cumulus cells by gently flushing through a fine-bore mouth-controlled pipette. Oocytes were examined using Nomarski differential interference contrast optics. Oocytes with no visible GV were classified as oocytes with GVB and regarded as undergoing meiotic maturation. The %GVB, defined as percentage of oocytes undergoing GVB per total number of oocytes from each ovary, was calculated. The experimental groups consisted of 5 to 12 perfused ovaries (Table 1). Data are presented as mean ± SEM.
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Follicular Fluid-MAS and Cholesterol Solutions
In the rat oocyte study, dilutions of FF-MAS were prepared from a stock solution containing 1 mg FF-MAS (molecular weight 412 g/mol) dissolved in 1 ml ethanol. In order to get a 0 µM (control group), 1 µM, 3 µM, 10 µM, or 30 µM FF-MAS use concentration we added 0 µl, 0.17 µl, 0.52 µl, 1.72 µl, or 5.16 µl of FF-MAS stock solution, respectively, together with 5.16 µl, 4.99 µl, 4.64 µl, 3.44 µl, or 0 µl ethanol, respectively, to give a final volume of 400 µl IBMX-medium (culture medium). The final ethanol concentration in all FF-MAS culture media, including the control group, was 1.29%.
In the ex vivo perfusion study of the rat ovary dilutions of FF-MAS and cholesterol were prepared from a stock solution containing 4 mg FF-MAS, respectively, dissolved in ethanol. In order to get a 0 µM (control group), 10 µM, 30 µM, or 60 µM FF-MAS use concentration we added 0 µl, 171 µl, 225 µl, or 450 µl of FF-MAS stock solution, respectively, together with 450 µl, 279 µl, 225 µl, or 0 µl ethanol, respectively, to give a final volume of 70 ml perfusion medium. In order to get a 60 µM cholesterol use concentration 450 µl of cholesterol stock solution was added to give a final volume of 70 ml perfusion medium. The final ethanol concentration in the perfusion medium of the cholesterol group and all FF-MAS groups, including the control group, was 0.64%.
Statistical Evaluation
In the rat oocyte assay and in the ex vivo perfusion model of the rat ovary the percentages of oocytes showing GVB were analyzed by one-way ANOVA followed by pairwise comparison (Students t-test) between treatment groups and untreated, negative controls. For the time course in vitro study the FF-MAS group and the spontaneously matured group were compared with the IBMX group. P < 0.05 indicated a statistically significant higher %GVB in treated groups compared to the negative control.
RESULTS
Effect of Hx and IBMX on Meiotic Maturation in the Rat Oocyte
The spontaneous meiotic maturation observed in CEO and DO after 22 h could not be blocked by 4 mM Hx (Fig. 1). In contrast, IBMX was able to dose-dependently inhibit resumption of meiosis (Fig. 1). The highest concentration of IBMX (250 µM) was able to inhibit spontaneous maturation significantly, whereas 5 µM and 50 µM IBMX were not effective.
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Effect of FF-MAS on Meiotic Maturation in the Rat Oocyte
Follicular fluid-MAS was able to overcome the inhibitory effect of 250 µM IBMX (Fig. 2). A clear dose-dependent response of meiotic maturation on both CEO and DO was observed with no discernible effect at the lowest test concentration (1 µM) of FF-MAS. Synthetic FF-MAS exerted significant stimulation on GVB at 3, 10, and 30 µM. Maximal stimulation was reached at 10 µM and 30 µM FF-MAS in both CEO and DO.
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After culturing DO and CEO for different times, it became evident that 10 µM FF-MAS led to a sluggish induction of GVB (Fig. 3). Whereas spontaneously matured oocytes exhibited a significant increase in GVB already at 3 h, 10 µM FF-MAS showed a significant GVB at 14 h.
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Effect of FF-MAS in the Ex Vivo Perfused Rat Ovary
Eighteen to 44 oocytes were collected from each ovary in the different treatment groups (Table 1). By using the ex vivo perfused rat ovary model it could be demonstrated that FF-MAS in the presence and absence of IBMX was able to dose dependently play a role in the control of meiosis (Table 1). Whereas ovaries perfused with IBMX alone (negative control) or LH + IBMX remained immature and only occasionally displayed meiotic maturation, despite the presence of 0.64% ethanol in the culture medium, 30 µM FF-MAS + 200 µM IBMX and 60 µM FF-MAS + 200 µM IBMX as well as 30 µM FF-MAS (alone) and 60 µM FF-MASe (alone) were significantly able to overcome meiotic arrest (Table 1). No significant effect was observed at the lowest test concentration (10 µM) of FF-MAS in combination with 200 µM IBMX. The highest dose of FF-MAS together with IBMX led to a comparable increase in GVB as could be observed in the positive oLH control group (Table 1). Conversely, 60 µM cholesterol + 200 µM IBMX were not able to significantly induce meiotic maturation (Table 1).
DISCUSSION
The rat assay data presented demonstrate for the first time that micromolar concentrations of synthetic FF-MAS are able to induce resumption of meiosis in a dose-dependent manner in IBMX-arrested rat oocytes. This effect can be observed when CEO and DO are cultured in the presence of FF-MAS. This finding is in accordance with earlier studies utilizing mice oocytes [14, 15] where FF-MAS was shown to trigger the resumption of meiosis in both CEO and DO. It has been substantiated that the inhibition of oocyte maturation is primarily mediated by the surrounding cumulus and granulosa cells, and that the relief of inhibition induced by LH or several growth factors, e.g., the epidermal growth factor (EGF), is a result of their effect on the cumulus cells and not on the oocyte itself [21, 22]. However, the most likely explanation for our data with FF-MAS is that FF-MAS exerts its meiosis-stimulating effect not via cumulus cells but by a direct interaction with the rat oocyte itself. However, it appears that cumulus cells are involved in the production and secretion of FF-MAS necessary for resumption of meiosis in DO [23]. To date the precise mechanism of how FF-MAS actually induces meiotic maturation remains unknown. Furthermore, the question of how FF-MAS can overcome the meiosis-arresting pathway of inhibitors (e.g., purines) in the follicular fluid is not presently known. Recent findings suggest that, in contrast to spontaneous maturation, FF-MAS-induced meiotic maturation requires protein synthesis and that a G-protein binding receptor mechanism is involved in FF-MAS signaling [24].
As mentioned by other authors in the rat [25], the nonselective PDE inhibitor IBMX was shown to prevent resumption of oocyte maturation in both DO and CEO. In DO this effect may be explained by the blockage in the breakdown of cAMP, especially by inhibition of the oocyte-specific PDE type 3 [26] that leads to elevated intraoocyte cAMP concentrations. In addition to the blockage of oocyte PDE type 3 in CEO, the nonselective PDE inhibitor IBMX, by augmenting granulosa cAMP levels transmitted to the oocyte [27], may be responsible for inhibiting oocyte maturation. Contrary to the mouse oocyte where comparatively low doses (5 µM) of IBMX were sufficient to prevent spontaneous meiotic maturation [15], 50-fold higher concentrations of IBMX (250 µM) were necessary to prevent spontaneous maturation in rat oocytes in vitro. Similarly after a culture time of 22 h, Hx, in concentrations known to inhibit meiotic maturation in the mouse (4 mM) [28, 16], were ineffective in blocking spontaneous maturation significantly in the rat oocyte. This is in contrast to in vitro observations by Törnell et al. [29] who showed that lower doses of Hx (0.5 mM and 2 mM, respectively) were able to inhibit GVB in rat CEO within 3.5 h. However, although significant, this inhibition was very low and reversible, as after 24 h nearly all CEO underwent GVB. Most likely, a transient arrest occurs that cannot be observed after 22 h with 4 mM Hx. Nevertheless, it should be cautioned that oocytes from different species may exhibit variable susceptibilities to putative inhibitors such as Hx. Indeed, Racowsky [30] found that, contrary to the mouse, the rat oolemma does not possess sufficient functional subunits of adenylate cyclase to stimulate adequate cAMP accumulation to maintain meiotic arrest.
Time course studies in rats and in mice [16] showed a discrepancy in the timing of GVB between spontaneous and FF-MAS-induced GVB. Compared to spontaneous induced GVB that reaches its maximum after 3 h in the rat, FF-MAS needs 14 h to induce significant resumption of meiosis. This indicates that spontaneous and FF-MAS-induced maturation occurs by different mechanisms. So far we do not know the stimulatory signals for FF-MAS-induced oocyte resumption of meiosis. Presumably, the mechanism of FF-MAS-induced meiotic maturation is mediated through a receptor protein, although the putative FF-MAS receptor has not yet been identified. Inhibition of transcription does not inhibit FF-MAS-induced meiosis in the mouse, which makes it unlikely that a nuclear receptor is involved in the cellular response to FF-MAS [24].
In the present study we have used a well-characterized model for ex vivo perfusion of preovulatory rat ovaries [20] to examine the effects of FF-MAS on oocyte maturation. We found that FF-MAS, the naturally occurring intermediate in the biosynthesis of cholesterol from lanosterol [31], was able to induce resumption of meiosis. In addition, FF-MAS was able to overcome IBMX-induced meiotic arrest. The sterol cholesterol, however, was unable to induce meiotic maturation in the presence of IBMX, indicating a specific role of FF-MAS in resumption of meiosis in the rat.
Similar to the in vivo situation, exogenous LH, in the absence of IBMX, stimulated oocytes to resume meiotic maturation in the ex vivo perfused rat ovary. The presence of mature oocytes after LH treatment is in line with earlier studies [32]. In fact only immature oocytes are found in LH-treated ovaries when perfusions are carried out in the presence of IBMX, indicating that this PDE inhibitor, most probably via cAMP increase, arrests the normal LH-induced stimulation of meiosis in our investigations. Given the fact that FF-MAS can overcome the IBMX-induced meiotic arrest, thereby showing opposing effects to these of LH + IBMX, it may be concluded that LH and FF-MAS act on meiosis by two different mechanisms. Although we do not know how to explain this difference so far, it may well be that FF-MAS acts downstream of the cAMP and protein kinase A pathway, thereby bypassing the inhibitory action of IBMX.
By comparing FF-MAS treatment with LH treatment in the absence of IBMX we got a significant increase in GVB in both treatment groups. One possible explanation for these ex vivo perfusion data could be that FF-MAS acts as a mediator of gonadotropin action. In the immature mouse ovary FF-MAS is present in low levels but accumulates within a few hours in response to an hCG stimulus [33]. In addition, there is solid evidence that a transient sixfold elevation of sterol cytochrome P450 14-demethylase, CYP51, occurs in rat ovaries after gonadotropin stimulation with eCG [34]. The highly conserved CYP51 is known to convert lanosterol to FF-MAS. The human and rat CYP51 are identical to 93% [35], which indicates that CYP51 plays a role in the gonadotropin-dependent formation of FF-MAS and provides a clue to understanding the mechanism of how gonadotropins induce meiotic maturation.
We have tested the action of FF-MAS on resumption of meiotic maturation both in vitro and ex vivo using well-characterized rat models. Comparable to observations with mice oocytes [14, 15] effective FF-MAS concentrations administered to achieve meiotic maturation in rat oocytes in vitro and in ex vivo could be found in the µM range. Although these concentrations seem to be high compared with sex steroids acting in the nM range, the sterol cholesterol at 60 µM was ineffective in inducing GVB in rats oocytes. Therefore, despite high concentrations, resumption of meiosis has to be regarded as a specific FF-MAS effect. Follicular fluid-MAS, like many other sterols, are highly sticky and lipophilic molecules [17] exhibiting extremely poor water solubilities. We have to keep in mind that in spite of an optically clear FF-MAS solution not all the FF-MAS is completely dissolved and may stick to plastic tubes, especially in the ex vivo perfusion system. Only a fraction of FF-MAS may reach the oocytes in the perfusion chamber after passage through the ovarian vessels, the interstitium and the different layers of the follicular wall. Follicular fluid-MAS was shown to be present at high concentrations (1.6 µM) in individual follicular fluids from women undergoing in vitro fertilization treatment [36]. It is therefore proposed that µM doses of FF-MAS do not necessarily represent pharmacologically active but more likely physiologically active concentrations. However, more detailed studies of FF-MAS concentrations in culture solutions and the ex vivo perfused ovary are required.
In summary, our results from well-characterized rat models in vitro and ex vivo demonstrate that FF-MAS is an effective mediator in the resumption of meiosis in the rat. Based on the different effects observed comparing spontaneous with FF-MAS-induced meiotic maturation in vitro and LH + IBMX with FF-MAS + IBMX-induced maturation ex vivo, we propose that FF-MAS mediates resumption of meiosis by a new, so far unknown mechanism. Although, the precise mechanism of FF-MAS action remains to be clarified, our data support the view that FF-MAS, beyond its effect in vitro, has the potential to induce resumption of meiosis in situ.
ACKNOWLEDGMENTS
We cordially thank Lam-Quoc Cam for his expert technical assistance.
FOOTNOTES
First decision: 21 January 2000.
1 Correspondence: Christa Hegele-Hartung, FC/HT Research of Schering AG, Müllerstrasse 170-178, D-13342 Berlin, Germany. FAX: 49 30 46818056;christa.hegelehartung{at}schering.de 
Accepted: September 7, 2000.
Received: December 20, 1999.
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